|Year : 2014 | Volume
| Issue : 4 | Page : 819-833
Radiation induced bystander effect and DNA damage
Nasir Jalal, Saba Haq, Namrah Anwar, Saadiya Nazeer, Umar Saeed
Department of Healthcare Biotechnology, Atta-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan
|Date of Web Publication||9-Jan-2015|
Department of Healthcare Biotechnology Atta Ur Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad 44000
Source of Support: None, Conflict of Interest: None
Bystander effects (BSEs) have been investigated for a long time but without much deliberation as to the cause in targeted cells and the subsequent effect in naïve cells. BSEs have traditionally been associated with radiation. Currently, this phenomenon is at a juncture where nuclear DNA damage is being debated as either essential or nonessential. If DNA damage is essential for the bystander signal (BSS) production then, this raises a number of questions about, radiotherapy and chemotherapy of cancer patients. This review presents a detailed analysis of the work done to investigate nuclear DNA damage versus exclusively cytoplasmic targeting with ionizing radiations and measurement of bystander end-points in naïve cells. The review also analyzes some of the research work done to investigate cell models that were developed specifically to study and track radiation-induced DNA damage to construct mutation spectra. Production of reactive oxygen species and reactive nitrogen species as possible candidates of the elusive BSS are also discussed besides the signal transduction pathways implicated in reception of a BSS by the naïve cell.
Keywords: Bystander effect, DNA, health, radiation
|How to cite this article:|
Jalal N, Haq S, Anwar N, Nazeer S, Saeed U. Radiation induced bystander effect and DNA damage. J Can Res Ther 2014;10:819-33
| > Introduction|| |
Radiation induced bystander effect (RIBE) is a condition in which cells that have not been directly targeted by ionizing radiation (IR) exhibit symptoms of exposure such as chromosomal instability, telomere aberrations, cell death and micronucleation  [Figure 1]. The effects of radiation on the cell are many and random. There are direct as well as indirect effects the latter of which are referred to as bystander effects (BSEs). Mutagens other than IR may also produce BSE. Direct effects usually results fromthe interaction of IR with all cell components while indirect effects result via a mediator, such as factors released into the medium surrounding exposed cells, which then signal to naïve cells causing DNA damage. Examples of direct effects of radiation include genomic instability, chromosomal rearrangements, delayed mutation, DNA nucleotide repeat instability, cellular transformation, and cell death. It is postulated that the same effects can be observed in naïve cells which are exposed to bystander signal (BSS) from targeted cells. It is still unknown what form or forms of damage to targeted cells actually leads to the generation of BSS. The hypothesis that this project investigates is that DNA damage also increases the mutation rate in directly targeted and nontargeted naïve cells. This is not to say that only DNA damage could cause BSE or is required in any way to initiate a BSE. Besides specific DNA damage, IR can cause damage to other cell components and may, therefore, up regulate BSE. This DNA damage could comprise base damage, single-strand breaks, double strand breaks (DSBs) or even the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) due to the ionizing effect. DNA damage response and repair of endogenous and exogenous sources of damage represent important defenses to prevent tumorigenesis. Hence, better understanding of the link between DNA damage and bystander mutagenesis will provide us with new strategies for cancer treatment.
Nagasawa and Little,  first demonstrated that targeting an α-particle microbeam to a few cells caused the neighboring non-targeted cells to show radiation induced sister chromatid exchanges (SCEs), the field of bystander mutagenesis has become a topic of much debate and research. Evidence shows that as well as direct DNA damage dependent effects; the radiation treated cells produce signals that are hereby referred to as "BSSs" that can affect the neighboring nonirradiated cells. This phenomenon has been called "BSE".  Ever since the discovery of BSE  it has been associated with variable doses of targeted nuclear radiation  and data were presented in favor of a nonnuclear (cytoplasmic) target of the phenomenon. It was argued , that DNA damage is not essential for BSS and that cytoplasmic targets could also produce the BSE. It has been re-affirmed that BSS is independent of DNA damage and DNA repair capacity of the irradiated cells.  This relatively new field of research is, therefore, at a critical stage, and it is important to determine whether DNA damage due to targeted nuclear radiation is the only source of BSS or not. The effects of radiation induced BSS on naïve cells are many and have been studied in detail. Using either precision microbeams or low-dose internal radiation β-particles in mammalian cells investigators were able to establish end-points in naïve mammalian cells, which are discussed in further detail later in this review. Direct evidence of BSE in humans does not exist, as yet, and there were few data available for in vivo systems. , In one such study tritiated thymidine labeled lymphocytes were transferred into the spleen of unirradiated control group of mice, but no change in apoptotic or proliferative capacity of spleen cells was observed. This does not in any way rule out the existence of BSEs in mammals generally and humans specifically; it just suggests that more research is required to understand the phenomenon and to evaluate the existence of BSS in mammals and humans.
| > Radiation Induced Bystander Effect and Its Implications for Cancer Treatment|| |
Observation of bystander end-points at low doses of IR, that is, <0.5 Gy  support linear no threshold hypothesis, stating there is no minimum safe dose of radiation. All types of radiation exposure are regulated according to this hypothesis and take into account radiation quality factors. There is no direct evidence for BSEs in humans  but three-dimensional artificial human tissue irradiations with helium ion  have demonstrated the existence of BSEs. This data suggests their existence in humans following radiation exposure. No pronounced γ-H 2 AX focal formation; an indicator of DNA damage  has been observed in cultured cells. The γ-H 2 AX foci normally appeared 30-40 min after irradiation of cultured cells and 12-48 h later in geometric human tissues followed by decrease over 1 week period.  Whole body nematode worm irradiations support existence of BSEs.  This study includes measurement of expression of green fluorescent protein chimera of a body stress gene heat shock protein-4 (hsp-4) in body regions without γ exposure.
Another approach for investigating BSEs used the implantation of cancer tissues in naïve mice. DNA damage in proliferative tissues distant from the implanted cancerous tissues suggested that BSEs operate in vivo. The BSE could have a detrimental impact on naïve cells making up proliferative tissues such as the gastrointestinal crypts and skin.  An inflammation cytokine chemokine (C-C motif) ligand 2/monocyte chemoattractant protein-1 was implicated in the process of this distant tissue DNA damage caused by cancer implants. Most RIBE are studied with regards to dose dependence, signal potency, signaling range, radiation source dependence, timing and cell type dependence.  BSEs are classified into three groups, namely (1) bystander effect, (2) abscopal effects and (3) cohort effect. Class 1 signifies radiation induced signal mediated effects in unirradiated cells, within the irradiated volume. Class 2 signifies radiation-induced effects in unirradiated tissues outside an irradiated volume and Class 3 signifies radiation-induced, signal mediated effects within the irradiated volume and between irradiated cells. There is a need to monitor the assumption that benefits of radiation for cancer treatment, outweighs risk. Radiotherapy of tumor cells may inadvertently cause them to release BSSs affecting the nontargeted cells to induce tumorigenic transformations.
| > Oxidative Stress for Bystander Signal Generation|| |
Oxidative stress is mainly caused by increased levels of reactive oxygen species (ROS) ROS in the environment of the cell. Oxygen derived free radicals can be generated by endogenous metabolism or exogenous sources such as IR, ultraviolet (UV) exposure, redox cycling drugs, and carcinogenic compounds.  Inflammation also leads to ROS and reactive nitrogen species (RNS) production, which are a hallmark of cancer hence, an important aspect of critical tumor progression.  ROS have been described as the energy landscape of a cell, to maintain normal homeostasis. Increased levels of ROS can cause DNA damage, as well as a whole range of cellular responses such as apoptosis, senescence, cell cycle arrest and even possibly cancer.  Increased levels of ROS can be generated in response to exposure of the cell to low levels of IR but they can also be helpful for a cell to avert the toxic effects of exposure (known as an adaptive response of a cell which makes the cell resistant to radiation upon subsequent exposures). It has also been argued that the long-term ROS effects can be damaging whereas the short-term effects of ROS may be protective. In the long-term, these increased ROS levels may cause proliferation of cells that is accompanied by accumulation of mutations.  ROS have been shown to form oxidative DNA adducts such as 8-hydroxguanine which in turn can lead to base modifications and transversions such as GC to TA; therefore, the oxidative state and hence (ROS) of a cell has been implicated as a possible BSS [Figure 2] that leads to the activation of stress response pathways, induction of proto-oncogenes, e.g. c-fos, c-Jun and c-Myc, and modified DNA base products that lead to DNA strand breaks, which ultimately are linked to an array of mutations, cell transformation and metastasis. ,
|Figure 2: A directly targeted cell receives damage from radiation but in turn produces reactive oxygen species (ROS) and reactive nitrogen species (RNS) which can affect the nontargeted cells. Such indirect effects of radiation have been argued as possible bystander signals (BSS) in medium. The possibility of these reactive species as BSS is controversial because of the very short half-life of ROS and RNS and the distance they can travel in medium as a result of diffusion is on a nanometer scale|
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Other stress-response studies carried out in targeted cells have shown that growth-related bystander response was observed in cells that received supernatants from α-particle irradiated cultures.  Such a response led to the upregulation of ROSin the bystander naïve cells, and it was mediated by the redox-potential activated tumor growth factor-β1 (TGF-β1) cytokine. These observations also showed that such a bystander response was associated with decreased levels of p-53 and p-21waf1.
| > Involvement of Cellular Environment in Bystander Signal Generation|| |
Activation or inactivation of signaling pathways involving tyrosine kinase, transcription factors, oxidation of cellular thiols and calcium homeostasis depends on the concentration of biological oxidants and thus the redox potential. , It was, hence, argued that the redox potential of the signaling pathways is a factor to consider in BSS. In studies involving the use of low-dose IR with β-particles, the antioxidant dimethylsulphoxide (DMSO) reduced the lethal effects in bystander cells produced by growing them with 3H-thymidine-labeled cells.  The argument was based on very short range β-particles (1 μm) emitted by tritiated thymidine. DMSO and lindane protected cells from BSE, by inhibiting both ROS and gap junction intercellular communication. Oxidative metabolism of γ-radiation targeted cells has also been investigated. ,, Use of antioxidants such as L-lactate and L-deprenyl  or drugs that inhibit the collapse of mitochondrial membrane potential , resulted in inhibition of the cytotoxic effects on bystander cells upon transfer from irradiated cultures. Chemical inhibition of BSS with antioxidants signifies it to be ROS based, while drugs that collapse mitochondrial membrane potential indicate the involvement of calcium ion signaling or components of apoptotic signaling as possible candidates of BSS.
| > Nitrogen Oxygen Species and Bystander Signal|| |
Nitric oxide is a biological signal and regulator in the human body. It generates nitrogen-oxygen species (NOS) upon reaction with superoxide radicals. 
NO + superoxide radical → NOS
Study showed that inducible nitric oxide, produced by X-radiation targeted cells, mediated the accumulation of p-53 and hsp72 protein in wild-type glioblastoma cells, upon transfer of conditioned medium from X-irradiated glioblastoma cells expressing mutant p-53. This accumulation of proteins could be inhibited when medium was treated with NOS scavengers or a nitric oxide synthase inhibitor.  The role of oxidative stress mediators in abscopal effects was found upon observing cytotoxic effects at distant sites from irradiation targeted solid tumors such as hepatocellular carcinomas.  It was argued that such abscopal effects were mediated by redox-potential sensitive cytokines. BSS generating specific factors or signaling mechanisms are still unknown.
| > Nuclear DNA damage for bystander effect-to be or not to be?|| |
A recent controversy  has surfaced, relating to the topic of whether or not nuclear DNA damage is essential for the BSE and if so, how could it impact the current radiotherapy intervention for cancer patients. Most particle radiotherapy focuses the depth deposition of dose to the actively dividing cells of a tumor mass. Calculations for dose depth deposition are based on the stopping power of radioactive particles in water as governed by the Bragg's peak principle.  This model is reasonably accurate, and the measured stopping of ions in compounds deviates <20%. The depth-dose deposition is measured mainly by calculating the stopping power of electrons/photons/protons emitted by the particle beam. Since most of these measurements are done in water,  assuming that human body is 90% water, the exact dose deposition traversing through the living tissue is rarely achieved. Ways to reduce the error have been discussed although radiotherapies still inadvertently damage healthy cells surrounding the tumor mass.  Stopping and range of ions in matter (SRIM) software developed by Ziegler (2004)  is constantly upgraded with up to 25,000 stopping values added until now. The software corrections made to the experimental calculation of stopping power of ions in compounds have been reported with the maximum accuracy of 89% of helium ions, compared to their last SRIM program of 1998. There is an 11% chance of error. Hence, the 20% error from Bragg's rule combined with 11% from SRIM, suggests that radiotherapies and radiation exposure can still cause damage to DNA of healthy cells besides targeting the tumor mass.  To add suspense to the elusive mystery, release of ATP has also been added to the realms of BSS.  It was recently reported that low dose γ radiation targeting causes the release of ATP from cells and that this signaling molecule is important for induction of a radiation stress response.
It has also been argued that the direct DNA damage from targeted IR can result in the production of oxidative stress, which could be responsible for the generation of BSSs. ,, Researchers evaluated the yield of single and DSBs resulting from low and high-linear energy transfer (LET) radiation exposures. In experiments using irradiated plasmid DNA, in the presence and absence of endonuclease III enzyme (which recognizes base modification and introduces a DNA break for base excision) they measured total single and DSB damage incurred by radiation. The authors argue that IR-induced DNA damage is predominantly produced by radiation mediated OH-radicals, an indication that oxidative stress is involved. Prise et al. (1999)  estimated that low-LET γ-radiation could give a DSB yield of up to 6.3 × 10−8 Gy/base pair. High-LET radiation could cause an additional 9% DSBs, and use of endo III enzyme could further raise the yield by 2.5%. There was no effect of the addition of endo III on background levels of single or DSB yield in the control group. Milligan et al. (2000)  confirmed these findings and both groups agreed on the involvement of clustered DNA damage being caused by OH-radicals. They used scavengers to reduce additional damage from these free radicals. Both teams also acknowledged that high-LET radiations cause more DNA damage compared to low LET. Rare cutting restriction enzymes were used to get small fragments of DNA that could be separated by pulse field gel electrophoresis (PFGE).  PFGE was then used to compare fragments of DNA resulting from varying doses of targeted IR. Almost 40 DSBs were produced in one P3 human epithelial teratoma cell by 1 Gy of X-rays. Induction of DSBs had a linear relationship with dose.
If free radicals are a by-product of radiation-induced DNA damage, then recent findings of free radicals and ROS as being possible candidates of BSS could be explained logically. ,,, The rationale for implicating DNA DSBs as responsible for the biological effects of radiation is that these effects are not caused by auto-oxidation. The traditional biological effects include multiple radicals reacting on opposite DNA strands.  Radiolytically generated OH-radicals form a radical with deoxyribose, but steric considerations make it unlikely to cause a break on the adjoining strand of DNA; hence single-strand breaks are more logical in this scenario. A DSB can potentially be caused upon action of these radicals on opposite strands.  Ward, ( 1981)  also reasoned that DSBs induced by high-LET radiation are an "attractive candidate" for investigating the production of BSS. However, he questioned the ability of one DSB being able to generate the BSS. He also proposed three sources of BSS generation from radiation targeting:
- A hydrated electron
- The ability of low-energy electrons to induce single or DSBs and
- Radiation-induced damage caused at sites that are otherwise inaccessible for species that cause auto-oxidative damage.
The ability of radiation targeted human keratinocytes to produce BSS for apoptosis in non-targeted naïve cells was tested.  Medium transfer from irradiated to unirradiated cells caused the appearance of characteristic early apoptotic end-points in naïve cells. No dose response for BSS generation by cells that were directly targeted by 0.5-5 Gy of radiation using a cobalt 60 source has been reported.
Another school of thought argues that for BSS production, nuclear DNA damage is not essential. To test this hypothesis of BSS Shao et al. (2004)  used precision microbeams to specifically target the cytoplasm of radioresistant T98G glioma cells (grown either singly or in co-culture with normal human fibroblasts AG01522) with charged helium ions (3He 2+ ). Even when one-helium ion specifically traversed through the cytoplasm of one-glioma cell, bystander responses were induced in the neighboring nontargeted glioma or fibroblast cells. Having used micronucleation as the end-point for bystander response in naïve cells, the team measured a 36% increase of micronuclei in glioma cells versus 78% increase in normal fibroblasts. They also demonstrated inhibition of bystander response upon treatment of cell populations with 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin. Inhibitors used are scavengers of nitric oxide hence suggesting the involvement of RNS as possible candidates of BSS. The team concluded that direct DNA damage is not essential for BSS and that the whole cell should be considered a sensor for radiation damage. The system used  raises some questions, for example, the fact that p53 mutant glioma cells were only traversed by helium ions to generate BSS and not normal human fibroblasts. The group used Nile red stained cells to avoid hitting the nuclei and set coordinates with 2 μm precision, to target one-helium ion to traverse the cytoplasm. If the cytoplasm was indeed targeted, we cannot rule out mitochondrial DNA as a target, hence becoming the source of extranuclear DNA damage for BSS production. Further investigation is, therefore, required to test if DNA damage is the source of BSS production.
The third aspect of this paradigm is if DNA repair has a role in BSS. DNA damage induced BSS production may not be affected by DNA repair, since repair could take hours; while BSS production is immediate, reaching its maximum within an hour of radiation exposure. In fact, DNA DSB were found as soon as 2 min after irradiation exposure in an experiment  while BSE in terms of γ-H 2 AX foci could be detected almost 30 min after medium transfer to naïve cells. It has been demonstrated that activation of BSS in DNA repair deficient xrs5 cells is independent of DNA repair capacity.  Researchers irradiated Chinese hamster ovarian (CHO) cells and separated the conditioned BSS containing medium later to apply to repair deficient xrs5 cells. They reported no increase in micronuclei in the naïve bystander repair deficient cells and reasoned that activation of BSS is independent of the DNA repair capacity of irradiated cells. The investigation sheds some light into the fact that DNA damage and repair capacity are independent of each other and suggests that BSS generation could precede the repair process.
For BSS experiments involving medium transfer it is important to maintain experimental conditions, cell type and cell cycle phase.  Hei et al. (2008)  argued that the extranuclear and extracellular events contribute to the overall low dose RIBE. Radiation-induced BSSs can be free radical based, and could be suppressed with DMSO.  However, this signal can vary under variable conditions and can activate different signal transduction pathways in the naïve cells.  Prise and O'Sullivan (2009)  discussed the implications of radiation-induced BSS vis-à-vis cancer therapy. They reasoned that the role of radiation-induced bystander response has to be in the context of our knowledge of cells communication and molecular pathways and targets outside the directly exposed fields could give a cumulative response to the radiation therapy.
If radiation independent DNA damage (DSBs to be specific) also produces bystander mutagenesis then it could have implications for cancer therapy since drugs such as alkylating agents function by disrupting DNA structure and creating DSBs.  As most of these drugs are not targeted to the tumor mass, they must have noncancer cell targets, receiving DNA damage, and possibly setting up a cascade of BSS events.
| > Nature of Bystander Signal|| |
The nature of BSS can vary according to the experimental model, treatment conditions, culture conditions and even the phase of cell cycle.  Following is an account of some of the possible candidates of radiation-induced BSS. Since these have been widely implicated in RIBE, these signals may have a role to play in radiation independent BSEs.
Tumor growth factor-α, tumor growth factor-β, tumor necrosis factor-α cytokines and reactive oxygen species
Although radiation exposure may trigger one pathway in a specific cell type, it does not mean that the same pathway would be triggered in different cell type.  Radiation can, however, trigger multiple pathways in a cell. Mitogen-activated protein kinase (MAPK) pathway signaling has been associated with growth factor-mediated regulation of such diverse cellular functions as proliferation, differentiation, senescence and apoptosis. Release of factors such as TGF-α, TNF- α and ROS from irradiated cells activate MAPK, extracellular signal-regulated kinase (ERK1/2), MEK1/2 and cyclooxygenase-2 (COX-2) pathways  in the naïve cells that received conditioned medium. TGF-β release from irradiated cells activates reduced nicotinamide adenine dinucleotide phosphate NAD (P) H oxidase and has been reported to cause the formation of γ-H 2 AX foci in naïve cells that receive conditioned medium. TGF-β causes suppression of proliferation and differentiation in lymphocytes, cytolytic T-cells, natural killer cells and macrophages, thus preventing immune surveillance of a growing tumor mass. 
An IR dose-dependent release of TGF-α in DU145 xenografts was measured by Hagan et al. (2004)  who demonstrated that this factor acts as a BSS stimulating the growth of naïve cells. The role of TGF-α in prostate cancer was also highlighted  in palliative cancer therapy, which puts into the picture the fact that if BSS is a result of radiotherapy in cancer patients, then it is all the more important to know its nature and dynamics. Once irradiated the xenografts produce TGF-α through the first wave of ERK1/2 activation indirectly targeted cells, while the same cytokine in medium when applied to naïve cells activated the second wave of ERK1/2 signaling cascade.
Apoptosis is a major end-point of bystander induced cells, while ROS was found involved in decreased mitochondrial membrane potential and increased intracellular Ca +2 levels.  Chelation of calcium and blockage of voltage-dependent Ca +2 channels suppressed apoptosis induction in bystander cells. Hei et al. (2008)  suggested a unifying model to explain the possible pathways involved in RIBE [Figure 3].
Cytoplasmic radiation targeting can also produce a BSS thus showing nuclear DNA damage is not required. ,, However, it is still possible that DNA damage, arising from mitochondrial DNA or intracellular calcium signaling could serve the same function in BSE.
| > Various End Points of Radiation Induced Bystander Effects in Mammalian Cells|| |
Although a wide variety of bystander end-points have been studied and often discussed en bloc for showing evidence to the existence of BSEs, they should not be mistaken for a singular/generic BSE.  Many biological systems have been shown to produce BSE under different set of conditions, and thus, a variety of BSSs have been reported. The evidence does not in any way suggest that bystander end points are generic but just gives a summary of many that have been observed in various systems upon exposure to a different set of chemicals, irradiations, or environmental stresses. Two different cell types may trigger different BSS pathways upon the same kind of radiation exposure. 
Using either precision microbeams or low-dose internal radiation β-particles in mammalian cells various investigators were able to measure BSE by using these end-points of BSS in a variety of naïve mammalian cells:
- Genetic 
- Epigenetic 
- Lethal mutations and altered gene expression 
- Activation of signal transduction pathways ,
- γ-H2AX foci formation 
- Delayed Apoptosis ,
- Sister chromatid exchanges 
- Micronucleation ,
- Neoplastic transformation in nontargeted cells. 
One of the earliest investigations into RIBE showed that the irradiation of targeted cells results in surviving colonies or clones that demonstrated delayed lethal mutations, which could be measured in terms of plating efficiencies.  In a separate investigation number of lethal mutations and delayed genetic mutations at the hprt locus among the progeny of cells from CHO cells and BALB/3T3 mice was measured. Reduced cloning efficiency was measured for up to 30 population doublings after X-ray exposure and delayed mutations at the hprt locus were recorded after 6-7 population doublings. 
The explanation into how delayed mutations were inherited remains unclear. However, there can be two possible explanations:
- Unrepaired DNA damage is passed on to the daughter cells for many generations until the damage becomes a heritable mutation
- Radiation exposure leads to a process that causes persistent elevation in background spontaneous mutations.
Delayed effect of a fractionated dose of radiation on bystander cells was also investigated in vitro and the results have been shown to be important for human patients receiving fractionated doses for radiotherapy. Although RIBE has been undoubtedly associated with low-dose radiation exposures, higher doses used for radiotherapy may also exhibit BSEs. HPV-G cells were given two doses of γ-rays separated by 2 h of recovery and conditioned medium removed from directly targeted cells was applied to naïve bystander cells. Survival of naïve HPV-G cells was low when conditioned medium was transferred from cells that were given a fractionated dose, compared to cells that were given one whole dose. This trend was surprisingly constant for over 1000-fold-range of doses from 0.005 Gy to 5.0 Gy.  And although most radiotherapy is carried out using fractionated doses, the research concluded that the BSEs of these radiotherapy doses must be further investigated. This important research has implication for an adaptive response of cells, where an initial priming dose imparts some radio resistance against a second much larger dose. Whether the first dose was lower than the second or higher or the same, the same BSE was observed on the survival of naïve cells, upon conditioned medium transfer. DNA damage responses following exposure to modulated radiation fields was evaluated.  A fibroblast cell line AG-1522B and prostate cancer cell line DU-145 was shielded 25, 50 and 75% from a dose of 1Gy to measure 53BP1 and γ-H 2 AX foci at 2mm intervals. The study reported an observation of out-of-field foci compared to the controls. The team also reported that this elevated residual DNA damage could be decreased following addition of nitric oxide synthase inhibitor; aminoguanidine. This data further provides proof in support of the hypothesis that ractive nitrogen species can in fact act as a potential bystander signal produced by irradiated cells.
In another investigation the adaptive response (where cells become resistant to the effect of radiation after first exposure) was investigated using the same cell line (HPV-G) and with separated conditioned medium as a priming dose to be applied to naïve cells.  Conditioned medium from cells irradiated at a higher dose was used as a challenge dose for naïve cells. Several end-points of RIBE that included ROS, apoptosis, calcium influx, loss of mitochondrial membrane potential, and calcium influx were examined. ROS were found to be involved in apoptosis while calcium influx varied in magnitude across the exposed cell population. No change in mitochondrial membrane potential was recorded for 0.5 Gy conditioned medium used as a primer and separated by a 24 h challenging dose of 5.0 Gy conditioned medium. This study concluded that HPV-G cells did show an adaptive response.
This account underlines the extent of complexity that this phenomenon can demonstrate using the same cell line, but looking at different end-points of BSS.
| > Spatial and Temporal Response of Radiation Induced Bystander Effects|| |
Very few experiments have been designed that measure the spatio-temporal kinetics of BSS. ,, Choosing an optimal time for the release of BSS into the surrounding medium is critical to ensure consistent measurement of a chosen end-point in the naïve cells. The peak time observed in some of these experiments  was 30 min after irradiation and used γ-H2AX foci as the end-point. However, DNA DSBs due to irradiation could be observed as soon as 2 min after exposure. The average level of DSB formation at 30 min after irradiating AG1522 normal human diploid fibroblasts was 3-fold indirectly targeted and 2-fold in naïve cells, compared to the control. The level of DSB remained steady for up to 6 h and then declined. The study also reported that medium containing the BSS could be transferred to anywhere in the petri plate (specially designed, having Mylar sheathing and aluminum shielding) to induce DSB in naïve (unirradiated cells). The percentage of DSB induced in naïve cells did not seem to depend on radiation dose delivered to the targeted cells. The team demonstrated the existence of temporal kinetics of BSS at least at very early stages of postirradiation and depended on the proximity of the naïve cells to the cells producing the signal. The team also showed that DMSO and lindane significantly inhibited the number of induced DSB in naïve cells, suggesting the involvement of ROS and gap junctional intercellular communication for BSS.
Zhang et al., (2009)  worked with WTK1 lymphoblast's and reported that maximum mutations at thymidine kinase (TK) locus were recorded 1 h after BSS containing conditioned medium was applied to naïve cells. It was also demonstrated that the directly targeted cells needed at least 1 h to generate sufficient BSS and that it diminished in 12-24 h. It is likely that radiolytically generated ROS are too unstable to diffuse to the naïve bystander cells and act as a potent BSS since they decay in nano seconds and diffuse over a distance of few nanometers. , In another unique investigation  a single cell was targeted with low-dose radiation and clusters of DNA damage were observed in naïve cells, as a function of BSE and data were showed that BSE was independent of the dose of radiation exposure. They were also able to establish a spatial response by observing transfer of BSS up to 3 mm away from the targeted cell and suggested a chain reaction that is set up by the one targeted cell to series of naïve cells. In a temporal and spatial kinetics investigation Hu et al. (2006)  demonstrated that BSS could travel as far as 7.5 mm away from the radiation targeted region, and that bystander end-points could be observed only 2 min after exposure of directly targeted cells and transfer of conditioned medium to naïve cells. They also demonstrated that the BSE remained stable for up to 6 h after exposure, but they argued that a time-dependent DSB response in bystander cells could be observed based on the distance of naïve cells from directly targeted cells.
Lyng et al. (2000)  used a p-53 null, immortalized human keratinocyte cell line to measure the increase in ROS in the medium of irradiated cells as a means to measure BSE. They found out that the increase in ROS continued for about 6 h after the medium was transferred from targeted cells to naïve ones. Zhang et al. (2009)  also investigated some of the temporal aspects of BSE while working with a p-53 mutant lymphoblast cell line WTK1. The team conducted kinetic studies and found out that it required up to 1 h generating sufficient signal to induce maximum level of mutations at the TK locus in the naïve cells that received conditioned medium from cells targeted by IR.
| > Saturation Effect of Radiation Induced Bystander Signal|| |
It has been argued in most medium transfer experiments that if the BSS is soluble in a medium then its dilution should decrease the effect on any end-point under consideration. Some experiments have been done to prove that point. In an experimental model cell system WTK1  it was shown that a 4-fold dilution of the conditioned medium (containing BSS) was required to render the medium ineffective for its bystander property; in contrast a 20-fold decrease in irradiated cell number (used to generate a BSS) was required to achieve the same dilution effect. The team also reported an adaptive response of cells which imparts a radiation resistant property to the targeted cells if primed with a low IR dose. Other interesting data were suggested that low levels of BSS exposure imparted an adaptive property to naïve cells also. Some researchers have investigated the effect of radiation dose on naïve bystander cells as a source of BSS reduction. Some experiments ,,, with -RIBE have shown convincing data were that a definite radiation exposure dose response could not be observed in naïve cells. The end points observed in these studies included micronuclei formation and chromosomal aberrations in bystander cells and their progeny.
| > Cell Models for Bystander Studies|| |
Several cell models have been developed to study DNA damaging effects of RIBE, most common of which is the lymphoblastoid cell line TK6. A mutation assay for TK locus of TK6 cell line was first developed by Liber and Thilly (1982)  and the locus characterized later by Grosovsky et al. (1993).  This human lymphoblastoid cell line comes from the spleen of a donor and has a doubling time of 12-16 h.  The 13.5 kb human TK1 gene is located on chromosome 17q23.2, oriented from telomere to centromere  and it is an autosomal gene that codes for a salvage pathway enzyme for nucleotide synthesis. TK6 is a heterozygous TK+/−cell line with frame-shift mutation in exons 4 and 7 of the inactive allele while a nine base pair deletion in exon 1 of the active allele was introduced in a cell line (TX528) derived from TK6. Most DNA DSBs in G1 phase are repaired by end-joining (EJ) while any leftover damage is repaired by homologous recombination (HR) in the postDNA replication phases of S and G2.  It has also been argued that EJ can operate throughout the cell cycle. Some modified TK6 cell lines have an I-Sce1 site engineered into the TK1 locus. This powerful tool enables one to track and study the DNA damaging effects of any DNA damaging agent. I-Sce1 endonuclease is a mitochondrial intron-encoded endonuclease of Saccharomyces cerevisiae which has a very low probability of cutting the DNA even within large genomes.
The first demonstration of using I-Sce1 as a tool to study genetic recombination in the mitochondria of yeast cells was done by Nakagawa et al. (1992).  Since then investigators have been inserting I-Sce1 restriction site into specially designed cell models for site-directed mutagenesis. The objective for this insertion has been to use it as a powerful tool of gene targeting specifically to induce DSBs using I-Sce1 restriction enzyme and studying the fate of this site directed DNA damage.  In separate investigations, it was demonstrated that DSBs can be initiated by the expression of I-Sce1 enzyme at predetermined location in a mammalian genome. , The probability of this sequence occurring twice in the human genome, other than the sequence inserted, is very low, and it was recently shown that only five off-target (nonspecific) sites for I-Sce1 exist in the human genome.  This model system can potentially mimic the radiation dependent DSB DNA damage that has previously been linked to bystander mutations in naïve cells.
Such a cell line using I-Sce1 insert in intron 4 of TK1 gene was constructed by Honma et al. (2003)  to identify the relative contribution of DNA repair using the two dominant DNA repair pathways of nonhomologous EJ (NHEJ) and HR. This cell line was derived from TK6 cells, in which the I-Sce1 insert was in intron 4, only 75 bp upstream of exon 5 in the active TK1 allele. Using this system it was reported  that 70% of all cells targeted for I-Sce1 induced DSBs were repaired by EJ which was also accompanied by deletions ranging from 100 to 4000 base pairs. In 23 out of 34 mutants analyzed for hemizygosity, the deletion size was more than 876 bp, while three out of 34 mutants had deletions of over 7.8 kilobases (kb). Molecular analysis of the remaining 30% cells showed that the repair caused complex DNA rearrangements like SCEs. It has been acknowledged by investigators  that the relative contribution ratio of EJ: HR of DSB repair may vary in mammalian cell lines, but EJ is the predominant mechanism. ,, As is logical and also affirmed by Honma et al. (2003)  the I-Sce1 system cannot recover every genetic consequence and is biased in favor of large sized deletions that include the nearest exon resulting in loss of heterozygosity; smaller deletions however that do not affect the TK function remain undetected because they will not be isolated as trifluorothymidine resistant mutants.
| > P-53 Regulation of DNA Damage Checkpoints|| |
The p-53 protein has been shown to play a pivotal role in DNA damage repair pathways, including DSBs, single strand breaks, base excision and mismatch repairs.  Following DNA damage, p-53 is phosphorylated and stabilized by its dissociation from MDM2, and going through posttranslational modifications it binds to DNA at target gene promoters that contain p-53 response elements.  In addition to the classical belief of p-53 being the guardian of the genome, a more collective aspect has been presented  that argues two definite roles as (a) guardian of genome and (b) policeman of oncogenes. The former role is activated by DNA damage stress signaling through ataxia telangiectasia mutated/ataxia telangiectasia mutated RAD 3 related (ATM/ATR) and checkpoint kinase 1/2 (chk1/2) kinases while the latter role depends on the p-53 stabilizing protein ADP-ribosylation factor (ARF). Given a cell that undergoes DNA damage and given the cell type, the presence of p-53 will determine entry of a cell into S-phase and its subsequent exit. The crucial observation that p-53 was either mutated or inactivated in almost 50% of human cancers  led to providing evidence in favor of a logical conclusion that if the cell does not have a cell cycle checkpoint, then it must be vulnerable to an override of p-53 independent checkpoints.  These logical conclusions were confirmed in two subsequent research papers , that demonstrated the use of caffeine to induce sensitization of p-53 deficient rat fibroblasts or human lung adenocarcinoma cells respectively to DNA damaging agents like IR. The p-53 independent checkpoints were argued to be operating at the G2-M transition mediated by the ATM/ATR-chk1/2-cell division cycle 25c (2-cdc25c)-cyclin B/cdc2 pathway.
| > Variable Levels Of P-53 Can Regulate Apoptosis or Cell Cycle Arrest|| |
Ceballos et al. (2005)  have reasoned that higher p-53 levels in human leukemia K562 cells can induce apoptosis while lower levels promote cells cycle arrest. These cells harbor a temperature sensitive allele of p-53, and the gene array analyses were compared with cells carrying wild-type p-53. The team successfully demonstrated that the c-Myc activation can impair p-53 mediated apoptosis by down regulating many downstream target genes of p-53 in leukemia cells that lack -ARF. On the other hand the cells expressing wild-type p-53 encoded chaperones hsp105, hsp90 and hsp27 that impart protection against programmed cell death. This research was important as to how apoptosis could be affected by p-53 levels in a cell. The team also looked at cancer cell lines derived from various tissues to validate their results and generalize the role of p-53. Two tumor cell lines that were developed from NonHodgkin's lymphoma were shown, through immunoprecipitation, to have mutant type p-53  and it was reasoned that p-53 mutation could indicate disease progression. However, (ARF) was shown to be mutated or repressed by promoter methylation in many lymphomas and tumors in general.  Ceballos et al. (2005)  have also shown that p-53 mediated cell cycle arrest is promoted by p-21 which inhibits cyclin dependent kinase. p-21, on the other hand, is inhibited by a c-Myc/Miz1 heterodimer complex and that this complex is required for c-Myc mediated repression of many genes besides p-21. A c-Myc/Max complex on the contrary upregulates certain genes upon binding with specific DNA sequences called (E-boxes).
| > DNA Damage and Repair in Radiation Targeted Cells|| |
The cells that are directly targeted by radiation produce a BSS. It is assumed that this induced DNA damage may be involved in the production of a BSS.  When factors that regulate DNA repair were inhibited (through RNA interference) it was shown  that the p-53 status of human lymphoblasts (WTK1 p-53 mutant, TK6 p-53 wild, NH32 p-53 null) did not affect either the production or reception of radiation-induced BSS. It was also demonstrated  that inhibition of DNA-dependent protein kinase (DNA PK) (one of the major enzymes involved in NHEJ repair) caused the mutation fraction of directly irradiated cells to increase in WTK1 cells, but decrease in TK6 and NH32 cells. However, DNA PK inhibition led to increased mutation fraction in bystander cells, regardless of the p53 status of a cell line. This suggests that the reception of BSS by naïve cells is independent of p-53 status, but the production of signal may have some p-53 involvement. Honma et al. (2003)  have also concluded through targeted DNA DSBs at an I-Sce1 site that NHEJ efficiently repairs most of the induced DSBs in DNA. If however, there is a misrepair of DNA DSBs; it becomes expressed as a mutation and sometimes large deletions cause mutations that could be lethal. Later studies conducted on the dose and time responses of lethal mutations and chromosomal instability due to IR  showed that there was an absence of lethal mutations in the descendants of HPV-G keratinocytes that received a low dose (0.5 Gy) of γ-rays, compared to cells that were given higher doses (1-3 Gy). The team also showed that when descendants of cells that were exposed to (0.5 Gy) α-particles were allowed to form colonies, their colony forming ability was reduced by 80%. The team did not rule out a relationship between lethal mutations and low LET radiations but argued that this relationship could be more complex. In a recent investigation  a team used mitochondrial DNA depleted (p 0 ) human small airway epithelial cell model in comparison with mitochondrial DNA proficient (p + ) cells. They targeted this cell model with a precision high LET microbeam to measure an increased autophagy, micronuclei formation, NFκB expression and an upregulated mitochondrial inducible nitric oxide synthase (iNOS) in P+ cells. This investigation demonstrated that mitochondrial DNA is essential for any high LET targeted oxidative DNA damage.
| > DNA Damage and Repair Dynamics in Bystander Induced Cells|| |
Reports of DNA damage in bystander induced cells have been appearing in the literature for some time now, but most of these studies are based on an indirect measure of the BSE like the phosphorylated H 2 AX foci formation. Sokolov et al. (2007)  reported the accumulation of DNA DSBs in bystander cells by measuring γ-H 2 AX focus formation. Another study  indicated that the additional occurrence of 1.3-2 foci on an average could account for the DNA DSBs in bystander induced normal human astrocytes and T98 glioma cells. The team also reasoned that since DMSO, filipin and anti-TGFβ-1 could inhibit the induction of γ-H 2 AX, therefore, membrane bound signaling could be involved.
Bystander induced cells showed induction of AP endonuclease 1, proliferating cell nuclear antigen and replication protein A (RPA). These factors point toward the participation of base excision repair (BER) for repairing the single-strand breaks in bystander induced cells.  The team, therefore, reasoned that BER inhibition should amplify the bystander induction. The authors have also discussed that bystander induced γ-H 2 AX foci formation involves ATR but not ATM or DNA PK, this would also indirectly confirm findings of Zhang et al. (2008)  who reported an increased mutation fraction in naïve bystander cells, when conditioned medium was transferred from DNA PK inhibited cells. It has been argued  that bystander induced cells generally show an up-regulation of p-53 and p-21 but a down-regulation of CDC2, cyclin B1 and Rad 51. A team recently reported  to have found no evidence of bystander induction in human fibroblasts by using carbon and uranium heavy ions to target single cells. They used γ-H 2 AX foci, SCEs and micronucleation as end-points.  The team argued that the results of the investigation did not exceed the experimental error values hence, they concluded that the BSEss are either lacking, or less pronounced.
| > DNA Repair Enzymes and Effect of Inhibition on Bystander Mutagenesis|| |
In the event of DNA damage many damage sensor enzymes are activated that trigger the repair process like ATM, ATR and DNA PK. In the bystander induced cells, the inhibition of DNA PK and ATM was reported to have no influence on the number of γ-H 2 AX foci, but a mutation of ATR could abrogate this induction.  It was interesting to note that chemical inhibitors were compared to the mutated form of ATR for a study, but as a matter of fact a commercially available ATR inhibitor does not exist. This investigation also reported that bystander focus formation was restricted to the S-phase of cell cycle. The foci were reported to persist in bystander cells for up to 72 h but 20 h postirradiation, showed the maximum induction. It is again important to mention that these end-points could vary with the experimental model and conditions. This is a matter of controversy in the field of -RIBE, however about γ-H 2 AX focal formation, it is clear that the time of appearance of foci can be from 2 min of post-exposure to 6 h.  Small molecules like CGK733 at low concentrations (600 nm) and high concentrations (10 μM) can be used to inhibit ATM or ATM/ATR in tandem respectively. Similarly, NU7026 (5 μM) for DNA PK  can be used. One compound NVP-BEZ235  for inhibiting ATR is still being tested for its efficacy.
| > Electroporation of Small Molecules and Plasmids Into Host Cells For Bystander Studies|| |
Electroporation is a highly efficient way of transporting small molecules and vectors into host cells via electrically induced pores in the membrane. , DNA is transported by electro diffusion in the presence of 1 mM Ca 2+ by pulse duration of 20-40 ms. Studies done in yeast cells have shown that electro diffusion of free DNA in the medium is 7 × 104 times more efficient than simple diffusion. The process involves cascade of events for transport of DNA into the host cells which includes pore formation, pore enlargement and transport of large molecules across the membrane.  The production of heat and free radicals during electroporation can be successfully inhibited using superoxide dismutase (SOD) and catalase.  Lipid peroxidation and hemolysis in red blood cells can also be induced during electroporation, depending on pulse field strength and duration. 
| > Electroporation Induced DNA Damage in Mammalian Cells|| |
Evidence suggests that the electroporation can cause DNA damage in the host mammalian cells by increasing applied voltage or capacitance.  DNA damage increased markedly at field strengths of over 400 V/cm, but that could be counteracted by lowering the capacitance from 1160 uF to 690 uF and temperature increase of about 7°C was observed at high voltage. Thus, it can be assumed that in restriction enzyme experiments structural loss of the protein may not be an issue. However, the team concluded that the electropermeabilization, unlike radiation-induced DNA damage, may be small. Naked DNA was also nicked by electroporation.  This suggests that electroporation-based transformation comes at the cost of limited DNA damage and possibly the production of some free radicals. In my experiments, these factors were considered, and data normalized to subtract the DNA damaging effects of electroporation.
| > Superoxide Dismutase Degrades Superoxide Radicals|| |
Superoxide dismutases are a class of metalloenzymes that are present in all oxygen using organisms and protection against the oxidative damage of superoxide radical anion (ȱ2 ) by reducing superoxides (ȱ2 ) to oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ). ,, Overexpression of SOD has been demonstrated to have anti-proliferative and anti-tumor effects in vitro and in vivo. Highly toxic by-products of cellular metabolism like ROS and hydrogen peroxide were successfully degraded in α-particle (0-10 cGy) bystander induced human diploid fibroblasts using 300 units/mL of SOD.  The team showed that SOD and catalase could significantly reduce the number of micronuclei in radiation-induced bystander cells, and they also down-regulated p-53 and p-21. SOD is inactivated (but not inhibited) by large concentrations of hydrogen peroxide (~15 mM) but it may even be protected to some extent by the catalase enzyme. 
During acute inflammatory responses, the elevated levels of pro-inflammatory cytokines enhance the deleterious effects of ROS.  If the nature of DNA strand break induced BSS is ROS then degrading of these superoxides with SOD, and catalase should lower the bystander mutant fraction (MF).
| > Bystander Effects Are Not Unique To Ionizing Radiation|| |
As already mentioned, BSSs can vary either by type of cells, treatment conditions or even the phase of cell cycle following is an account of some treatments other than radiation that can also produce a BSE. BSEs have also been demonstrated through the use of chemotherapeutic drugs  and photodynamic stress agents.  Some chemotherapeutic agents that are known to cause the production of BSEs are:
- Mitomycin C 
- Phleomycin 
- Paclitaxel. 
It is known that in attached cells the BSS is transmitted through gap junctions and in suspension cells this happens via soluble factors released into the medium  and that BSSs could be detected in the medium for up to 24 h after radiation exposure.
| > Bystander Induced Activation of Mitogen-Activated Protein Kinase Targets|| |
Evidence suggests , that radiation-induced BSSs can activate some downstream MAPK targets such as p90RSK which is a member of ETS oncogene family (ELK1), activating transcription factor 2 and COX-2. Some investigators have shown small signaling molecules to be involved in BSS. Some of these BSS amines have been reported to include 5-hydroxytryptamine (5-HT, serotonin), L-DOPA, glycine or nicotine, following IR exposure.  MAPK pathway activation has also been observed in bystander cells  and involvement of oxidative metabolism alleged as being a possible BSS. SOD and catalase were shown to significantly inhibit the number of micronuclei formed, and decreased activation of downstream stress targets like p-21 and p-53 in naïve AG 1522 cells. It was suggested that ROS derived from flavin-containing oxidase enzyme (presumably NADP[H] oxidase) were the main source of upregulation of oxidative stress proteins p-21 and p-53. Other proteins believed to be activated as a response to stress included, NF κB, Raf-1, ERK1/2, c-Jun, p-38 MAP kinase. At least ERK1/2 and p38 again indicate toward the involvement of the MAPK pathway.
| > Inhibition of Mitogen-Activated Protein Kinase Pathway and Bystander Signaling|| |
Börsch-Haubold et al. (1998)  have demonstrated that a small organic compound called PD 98059 at concentration of 20 μM can inhibit thrombin-induced activation of p42 MAPK and p38 MAPK. Azzam et al. (2002)  also demonstrated that several downstream signaling proteins of the MAPK pathway were activated in naïve cells that were exposed to conditioned BSS containing medium. Within 1 min of exposure to 5 cGy radiation, Raf-1, ERK½, JNK and p38 were notably phosphorylated (activated). It was also argued that since many downstream proteins of the MAPK pathway are activated by ROS therefore the team was successful in partially inhibiting ERK1/2 and JNK with 30 min SOD and catalase inhibition.
| > Mitogen-Activated Protein Kinase Activation Initiated by Small Guanosine Triphosphate-Binding Proteins|| |
G-protein coupled receptors (GPCR) are identified as one of the most common membrane proteins involved in the transmission of signals from extracellular environments to the cytoplasm.  MAPK proliferative pathways have been reported to show activation upon mitogenic stimulation through a GPCR dependent manner. -ERK is a downstream signaling protein whose enzymatic activity increases upon mitogenic stimulation and impeding its function leads to loss of cellular proliferation. It has also been reasoned that constitutive activation of proteins upstream of MAPK and of itself can lead to a transformed phenotype and tumorigenesis.  It was reported that MAPK activation is initiated by small guanosine triphosphate-binding proteins, including RAS. These activations however are short-lived and must be converted to long-lasting forms to participate in this activation cascade as reported by Wilkinson and Millar (2000).  Mansour et al. (1994)  have proposed that prolonged activation of MAPK kinase, can lead to activation of oncogenic proteins like Ras, Raf, Src and Mos. Constitutively active MAPK mutants were designed having 400 times the normal expression in wild-type cells showed many hallmarks of a tumorigenic effect like cell rounding and high-saturation density when expressed in HEK293 and NIH3T3.
| > Raf And Mek Plays A Role In Cancer Progression and Phosphorylates C-Myc|| |
There is also substantial evidence that validates the role of Raf and MEK in cancer progression and promotion of cancer growth.  Thus, MEK1/2 are prime candidates for inhibition in a DNA strand break induced BSE for example MAPK pathway has been successfully inhibited using small molecules like PD98059  and U0126.  There is some debate about the specificity of these inhibitors since they are phosphor inositide 3 kinase specific, and there are other important kinase enzymes that may also be affected or inhibited. A p-38 inhibitor PD98059 was used to investigate the role of MAPK pathway in DSB-ABE. McCubrey et al. (2007)  have identified c-Myc as one of the downstream signaling proteins of the MAPK pathway. According to them, ERKs enter the nucleus directly to phosphorylate many transcription factors including Ets-1, c-jun and c-Myc.
| > Extracellular Signal Regulated Kinase1/2 And P38 Kinases Phosphorylate P-53 At Serine 15|| |
In the MAPK cascade the downstream kinases ERK1/2 and p38 have been shown  to phosphorylate p-53 at serine 15 in response to UV radiation exposure of mouse epidermal cell lines (JB6). ERKs and p38 were reported to form a complex with p-53 after UV (B) irradiation of these cells and that abrogation of the function of ERKs and p38 with PD98059 or SB202190 resulted in abrogation of the p-53 phosphorylation. 
| > Telomeres and Bystander Effects|| |
Telomeres are made up of single-stranded repeating units of TTAGGG (in mammals) at the 3' end and maintained by telomerase into a double-stranded form to protect it from being recognized as a DNA damage site. The length of telomeres varies between species and with age. Human telomeres on an average are 10-15 kbp in length while mice telomeres could be as long as 50 kbp.
The structure of telomeric DNA remains same almost in all eukaryotes, and the evolution of telomeres was to organize genome linearly and to protect and replicate chromosome ends.  Telomere-DSB fusions can be studied as possible targets of BSE as Nuta et al. (2008)  and others , have looked at the RIBEs on the telomeres of telomerase immortalized foreskin fibroblasts BJ1-hTERT and reported that telomerase enzyme does not play any role in BSEs.
| > Telomerase and Cancer Therapeutics|| |
The ribonucleoprotein telomerase regulates the lengthening of telomere at each cell division. In recent years this enzyme has become a major target for cancer treatment since it is required for the immortalization of all cancer cells, including some cancer stem cells.  It appears that telomerase repression and tight regulation is a tumor suppressor mechanism although telomerase is not regulated by an oncogene.
| > Telomere Position Effect and Chromosome Healing|| |
Kulkarni et al. in 2010  used plasmid sequences to integrate them next to a telomere to demonstrate that mammalian telomeres can suppress gene expression which they termed as the telomere position effect. They also demonstrated that a 100-kb sub-telomeric region was highly sensitive to DSBs that could lead to chromosome instability, however, this condition could be rescued by the addition of a new telomere to the break, which the team referred to as chromosome healing. These concepts are important for this project because it entails two important factors for consideration of MF studies:
- Sub-telomeric region is essential to induce DSBs in the telomeric region
- A plasmid sequence could be inserted near the telomere to collect bystander MF data.
| > Can Suppress The Dna Damage Response|| |
In mammalian cells, the protection from being recognized as DNA damage sites is dependent on a protein complex called Shelterin that associates itself to the telomeric DNA and imparts protection against the DNA damage machinery.  Suppression of DNA damage at telomeres is mediated by two central phosphatidylinositol 3-kinase-related protein kinases called ATM and ATR.  ATM predominantly responds to DSBs while ATR responds to single-strand breaks. A faster acting, sequence nonspecific DNA damage sensor protein called RPA binds at the single-stranded DNA damage site and recruits ATR/ATR interacting protein complex. Once this activation is achieved ATR, like ATM, phosphorylates up to several kilobases of the histone variant called H2AX at serine 139.  Since this process is not sequence specific, and a lot of RPA exists in the nucleus, therefore, the DNA repair is fast. Presence of the 3'telomeric overhang, if or when exposed, as a DNA damage site can be quickly repaired.
| > Dual Role of Telomere Dysfunction In Tumors|| |
Somatic or stem cells that give rise to tumorous cells that have reduced telomerase activity face a critical erosion of telomeres. This can have two effects on tumor development: 
- Dysfunctional telomeres can activate a DNA damage response which inevitable leads to either apoptosis or senescence
- Repair mechanism at dysfunctional telomeres can lead to genomic instability by forming dicentrics, telomere double stand break fusions or telomere-telomere fusions.
Both these additive factors fuel transformation in the genome that can potentially lead to tumor development.
| > Discussion|| |
At present, a diverse range of end-points of BSE have been observed and measured in various cell lines of naïve cells and the same cell line can produce a variety of bystander end points. BSEs are not unique to radiation because they have been observed upon treatment with certain alkylating agents producing ROS or RNS, however ROS or RNS do not usually diffuse in solution beyond a few nanometers therefore they can be ruled out as possible candidates of BSS in medium transfer experiments. The DNA damage, as well as cytoplasmic targets, can produce the BSE, although some experiments have demonstrated the production of RIBE by avoiding nuclear DNA, but these experiments did not consider mitochondrial DNA as a potential target. BSS is not dependent on DNA repair capacity of the irradiated cells, and the nature of BSS is a dynamic property. The nature of BSS varies and depends upon various factors; it also depends upon the cell model. Various endpoints, as well as cell lines, have been established to study the function of BSE, and most important of those is the TK6 modified lymphoblastoid cell line TX528. Using these cell line models and end points the temporal function of bystander varies with chosen end points. P-53 is an integral factor for DNA damage repair as it controls the cell cycle entry and exit and the level of P-53 plays a role, when the DNA damage is involved in the production of BSS. BSEs can also be seen after electroporation which in turn can cause production of free radicals. BSEs have also been demonstrated through the use of chemotherapeutic drugs and photodynamic stress agents. Considerable evidence has been put forward to show that the elusive BSS follows a MAPK pathway and that inhibiting this pathway with small molecules can essentially inhibit the BSE in naïïve cells.
| > Conclusion|| |
Bystander effects have long been ascribed to low dose IRs, but recent evidence shows that RIBE can be associated with radiation-induced DNA damage. Experiments have been done to avoid nuclear DNA targeting, and still BSEs have been observed. There is a strong indication that cytoplasmic targeting can also produce BSE. However, if mitochondria were inadvertently targeted then mitochondrial DNA damage could have triggered a cascade of events that leads to BSS. As far as DNA damage is concerned with regards to the production of BSE more research needs to be done. At this point of bifurcation in these two schools of thought, it is important to emphasize that investigation into DNA damage, as a possible cause for BSS production, needs the attention of researchers in this field. A recent research  has outlined the presence significantly higher levels of DNA damage in the normal colonic mucosa following radiotherapy, which is a strong indication of RIBE in vivo. This brings into perspective radiation doses used for therapeutic purposes. Some cellular defense mechanisms such as active DNA repair impart protection against radiolesions but evidence exists where BSS may be released within 30 min of radiation-induced DNA damage. Hence radiation-induced bystander mediated secondary tumorigenesis could be a consequence in cancer patients, undergoing radiotherapy.
| > References|| |
Prise KM, Folkard M, Michael BD. A review of the bystander effect and its implications for low-dose exposure. Radiat Prot Dosimetry 2003;104:347-55.
Nagasawa H, Little JB. Induction of sister chromatid exchanges by extremely low doses of alpha-particles. Cancer Res 1992;52:6394-6.
Mothersill C, Stamato TD, Perez ML, Cummins R, Mooney R, Seymour CB. Involvement of energy metabolism in the production of ′bystander effects′ by radiation. Br J Cancer 2000;82:1740-6.
Shao C, Folkard M, Michael BD, Prise KM. Targeted cytoplasmic irradiation induces bystander responses. Proc Natl Acad Sci U S A 2004;101:13495-500.
Prise KM, Folkard M, Kuosaite V, Tartier L, Zyuzikov N, Shao C. What role for DNA damage and repair in the bystander response? Mutat Res 2006;597:1-4.
Kashino G, Suzuki K, Matsuda N, Kodama S, Ono K, Watanabe M, et al.
Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells. Mutat Res 2007;619:134-8.
Blyth BJ, Azzam EI, Howell RW, Ormsby RJ, Staudacher AH, Sykes PJ. An adoptive transfer method to detect low-dose radiation-induced bystander effects in vivo
. Radiat Res 2010;173:125-37.
Blyth BJ, Sykes PJ. Radiation-induced bystander effects: What are they, and how relevant are they to human radiation exposures? Radiat Res 2011;176:139-57.
Mothersill C, Seymour CB. Radiation-induced bystander effects - Implications for cancer. Nat Rev Cancer 2004;4:158-64.
Sedelnikova OA, Nakamura A, Kovalchuk O, Koturbash I, Mitchell SA, Marino SA, et al.
DNA double-strand breaks form in bystander cells after microbeam irradiation of three-dimensional human tissue models. Cancer Res 2007;67:4295-302.
Hu B, Wu L, Han W, Zhang L, Chen S, Xu A, et al.
The time and spatial effects of bystander response in mammalian cells induced by low dose radiation. Carcinogenesis 2006;27:245-51.
Bertucci A, Pocock RD, Randers-Pehrson G, Brenner DJ. Microbeam irradiation of the C. elegans nematode. J Radiat Res 2009;50 Suppl A: A49-54.
Redon CE, Dickey JS, Nakamura AJ, Kareva IG, Naf D, Nowsheen S, et al.
Tumors induce complex DNA damage in distant proliferative tissues in vivo
. Proc Natl Acad Sci U S A 2010;107:17992-7.
Halliwell B, Gutteridge JM. Free radicals in biology and medicine. J Pharm Sci 1986;75:105-6.
Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A. Cancer-related inflammation, the seventh hallmark of cancer: Links to genetic instability. Carcinogenesis 2009;30:1073-81.
Lehnert BE, Iyer R. Exposure to low-level chemicals and ionizing radiation: Reactive oxygen species and cellular pathways. Hum Exp Toxicol 2002;21:65-9.
Christen S, Hagen TM, Shigenaga MK, Ames BN. Chronic inflammation, mutation, and cancer. In: Parsonnet J, Hornig S, editors. Microbes and Malignancy: Infection as a Cause of Cancer. New York: Oxford University Press; 1999. p. 35-88.
Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: Role in inflammatory disease and progression to cancer. Biochem J 1996;313:17-29.
Iyer R, Lehnert BE. Effects of ionizing radiation in targeted and nontargeted cells. Arch Biochem Biophys 2000;376:14-25.
Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radic Biol Med 2000;28:463-99.
Gabbita SP, Robinson KA, Stewart CA, Floyd RA, Hensley K. Redox regulatory mechanisms of cellular signal transduction. Arch Biochem Biophys 2000;376:1-13.
Bishayee A, Hill HZ, Stein D, Rao DV, Howell RW. Free radical-initiated and gap junction-mediated bystander effect due to nonuniform distribution of incorporated radioactivity in a three-dimensional tissue culture model. Radiat Res 2001;155:335-44.
Lyng FM, Seymour CB, Mothersill C. Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis. Br J Cancer 2000;83:1223-30.
Lyng FM, Maguire P, McClean B, Seymour C, Mothersill C. The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects. Radiat Res 2006;165:400-9.
Rubanyi GM, Ho EH, Cantor EH, Lumma WC, Botelho LH. Cytoprotective function of nitric oxide: Inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392-7.
Matsumoto H, Hayashi S, Hatashita M, Ohnishi K, Shioura H, Ohtsubo T, et al.
Induction of radioresistance by a nitric oxide-mediated bystander effect. Radiat Res 2001;155:387-96.
Ohba K, Omagari K, Nakamura T, Ikuno N, Saeki S, Matsuo I, et al.
Abscopal regression of hepatocellular carcinoma after radiotherapy for bone metastasis. Gut 1998;43:575-7.
Kojima S. Involvement of ATP in radiation induced bystander effect as a signaling molecule. Yakugaku Zasshi. 2014;134:743-9. ONLINE ISSN: 1347-5231.
Bragg WH, Kleeman R. On the alpha particles of radium and their loss of range in passing through various atoms and molecules. Philos Mag 1905;10:318.
Siiskonen T, Kettunen H, Peräjärvi K, Javanainen A, Rossi M, Trzaska WH, et al
. Energy loss measurement of protons in liquid water. Phys Med Biol 2011;56:2367-74.
Ziegler JF. Nuclear instruments and methods in physics research section B: Beam interactions with materials and atoms. SRIM 2004;219:1027-36.
Trikalinos TA, Terasawa T, Raman SI, Lau J. Particle Beam Radiation Therapies for Cancer. Technical Brief No. 1.(Prepared by Tufts Medical Center Evidence-Based Practice Center Under Contract No. HHSA-290-07-10055). Rockville, MD: Agency for Healthcare Research and Quality; Revised November 2009. Available from: http://www.effectivehealthcare.ahrq.gov/reports/final.cfm
[Last accessed on 2014 Aug 1].
Milligan JR, Aguilera JA, Paglinawan RA, Ward JF, Limoli CL. DNA strand break yields after post-high LET irradiation incubation with endonuclease-III and evidence for hydroxyl radical clustering. Int J Radiat Biol 2001;77:155-64.
Milligan JR, Aguilera JA, Nguyen TT, Paglinawan RA, Ward JF. DNA strand-break yields after post-irradiation incubation with base excision repair endonucleases implicate hydroxyl radical pairs in double-strand break formation. Int J Radiat Biol 2000;76:1475-83.
Prise KM, Pullar CH, Michael BD. A study of endonuclease III-sensitive sites in irradiated DNA: Detection of alpha-particle-induced oxidative damage. Carcinogenesis 1999;20:905-9.
Löbrich M, Ikpeme S, Kiefer J. Measurement of DNA double-strand breaks in mammalian cells by pulsed-field gel electrophoresis: A new approach using rarely cutting restriction enzymes. Radiat Res 1994;138:186-92.
Gudkov SV, Garmash SA, Shtarkman IN, Chernikov AV, Karp OE, Bruskov VI. Long-lived protein radicals induced by X-ray irradiation are the source of reactive oxygen species in aqueous medium. Dokl Biochem Biophys 2010;430:1-4.
Grosovsky AJ, Walter BN, Giver CR. DNA-sequence specificity of mutations at the human thymidine kinase locus. Mutat Res 1993;289:231-43.
Hei TK, Zhou H, Ivanov VN, Hong M, Lieberman HB, Brenner DJ, et al.
Mechanism of radiation-induced bystander effects: A unifying model. J Pharm Pharmacol 2008;60:943-50.
Dent P, Yacoub A, Contessa J, Caron R, Amorino G, Valerie K, et al.
Stress and radiation-induced activation of multiple intracellular signaling pathways. Radiat Res 2003;159:283-300.
Prise KM, O′Sullivan JM. Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer 2009;9:351-60.
Lieberman MW, Baney RN, Lee RE, Sell S, Farber E. Studies on DNA repair in human lymphocytes treated with proximate carcinogens and alkylating agents. Cancer Res 1971;31:1297-306.
Pardali K, Moustakas A. Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer. Biochim Biophys Acta 2007;1775:21-62.
Hagan M, Yacoub A, Dent P. Ionizing radiation causes a dose-dependent release of transforming growth factor alpha in vitro
from irradiated xenografts and during palliative treatment of hormone-refractory prostate carcinoma. Clin Cancer Res 2004;10:5724-31.
Seymour CB, Mothersill C, Alper T. High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation. Int J Radiat Biol Relat Stud Phys Chem Med 1986;50:167-79.
O′Reilly S, Mothersill C, Seymour CB. Postirradiation expression of lethal mutations in an immortalized human keratinocyte cell line. Int J Radiat Biol 1994;66:77-83.
Prise KM, Schettino G, Folkard M, Held KD. New insights on cell death from radiation exposure. Lancet Oncol 2005;6:520-8.
Azzam EI, De Toledo SM, Spitz DR, Little JB. Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures. Cancer Res 2002;62:5436-42.
Jamali M, Trott KR. Persistent increase in the rates of apoptosis and dicentric chromosomes in surviving V79 cells after X-irradiation. Int J Radiat Biol 1996;70:705-9.
Trainor C, Butterworth KT, McGarry CK, McMahon SJ, O′Sullivan JM, Hounsell AR, Prise KM. DNA damage responses following exposure to modulated radiation fields. PLoS One. 2012;7:e43326. doi: 10.1371/journal.pone. 0043326. Epub 2012 Aug 17.
Little JB, Gorgojo L, Vetrovs H. Delayed appearance of lethal and specific gene mutations in irradiated mammalian cells. Int J Radiat Oncol Biol Phys 1990;19:1425-9.
Mothersill C, Seymour CB. Bystander and delayed effects after fractionated radiation exposure. Radiat Res 2002;158:626-33.
Maguire P, Mothersill C, McClean B, Seymour C, Lyng FM. Modulation of radiation responses by pre-exposure to irradiated cell conditioned medium. Radiat Res 2007;167:485-92.
Staudacher AH, Blyth BJ, Lawrence MD, Ormsby RJ, Bezak E, Sykes PJ. If bystander effects for apoptosis occur in spleen after low-dose irradiation in vivo
then the magnitude of the effect falls within the range of normal homeostatic apoptosis. Radiat Res 2010;174:727-31.
Zhang Y, Zhou J, Baldwin J, Held KD, Prise KM, Redmond RW, et al.
Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects. Mutat Res 2009;671:20-5.
Hamada N, Matsumoto H, Hara T, Kobayashi Y. Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. J Radiat Res 2007;48:87-95.
Ward JF. Some biochemical consequences of the spatial distribution of ionizing radiation-produced free radicals. Radiat Res 1981;86:185-95.
Schettino G, Folkard M, Prise KM, Vojnovic B, Held KD, Michael BD. Low-dose studies of bystander cell killing with targeted soft X rays. Radiat Res 2003;160:505-11.
Ponnaiya B, Jenkins-Baker G, Brenner DJ, Hall EJ, Randers-Pehrson G, Geard CR. Biological responses in known bystander cells relative to known microbeam-irradiated cells. Radiat Res 2004;162:426-32.
Ponnaiya B, Suzuki M, Tsuruoka C, Uchihori Y, Wei Y, Hei TK. Detection of chromosomal instability in bystander cells after Si490-ion irradiation. Radiat Res 2011;176:280-90.
Ponnaiya B, Cornforth MN, Ullrich RL. Induction of chromosomal instability in human mammary cells by neutrons and gamma rays. Radiat Res 1997;147:288-94.
Liber HL, Thilly WG. Mutation assay at the thymidine kinase locus in diploid human lymphoblasts. Mutat Res 1982;94:467-85.
Honma M, Izumi M, Sakuraba M, Tadokoro S, Sakamoto H, Wang W, et al.
Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double-strand breaks in human cells. Environ Mol Mutagen 2003;42:288-98.
Nakagawa K, Morishima N, Shibata T. An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria. EMBO J 1992;11:2707-15.
Bellaiche Y, Mogila V, Perrimon N. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics 1999;152:1037-44.
Choulika A, Perrin A, Dujon B, Nicolas JF. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae
. Mol Cell Biol 1995;15:1968-73.
Petek LM, Russell DW, Miller DG. Frequent endonuclease cleavage at off-target locations in vivo
. Mol Ther 2010;18:983-6.
Essers J, van Steeg H, de Wit J, Swagemakers SM, Vermeij M, Hoeijmakers JH, et al.
Homologous and non-homologous recombination differentially affect DNA damage repair in mice. EMBO J 2000;19:1703-10.
Takata M, Sasaki MS, Sonoda E, Morrison C, Hashimoto M, Utsumi H, et al.
Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 1998;17:5497-508.
Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol 2007;8:275-83.
Riley T, Sontag E, Chen P, Levine A. Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 2008;9:402-12.
Efeyan A, Serrano M. p53: guardian of the genome and policeman of the oncogenes. Cell Cycle 2007;6:1006-10.
Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell 1992;70:523-6.
Powell SN, DeFrank JS, Connell P, Eogan M, Preffer F, Dombkowski D, et al
. Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay. Cancer Res 1995;55:1643-8.
Russell KJ, Wiens LW, Demers GW, Galloway DA, Plon SE, Groudine M. Abrogation of the G2 checkpoint results in differential radiosensitization of G1 checkpoint-deficient and G1 checkpoint-competent cells. Cancer Res 1995;55:1639-42.
Ceballos E, Muñoz-Alonso MJ, Berwanger B, Acosta JC, Hernández R, Krause M, et al.
Inhibitory effect of c-Myc on p53-induced apoptosis in leukemia cells. Microarray analysis reveals defective induction of p53 target genes and upregulation of chaperone genes. Oncogene 2005;24:4559-71.
Chang H, Benchimol S, Minden MD, Messner HA. Alterations of p53 and c-myc in the clonal evolution of malignant lymphoma. Blood 1994;83:452-9.
Krug U, Ganser A, Koeffler HP. Tumor suppressor genes in normal and malignant hematopoiesis. Oncogene 2002;21:3475-95.
Zhang B, Davidson MM, Hei TK. Mitochondria regulate DNA damage and genomic instability induced by high LET radiation. Life Sci Space Res (Amst). 2014 Apr 1;1:80-88.
Zhang Y, Zhou J, Held KD, Redmond RW, Prise KM, Liber HL. Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells. Radiat Res 2008;169:197-206.
Sokolov MV, Dickey JS, Bonner WM, Sedelnikova OA. Gamma-H2AX in bystander cells: Not just a radiation-triggered event, a cellular response to stress mediated by intercellular communication. Cell Cycle 2007;6:2210-2.
Burdak-Rothkamm S, Short SC, Folkard M, Rothkamm K, Prise KM. ATR-dependent radiation-induced gamma H2AX foci in bystander primary human astrocytes and glioma cells. Oncogene 2007;26:993-1002.
Fournier C, Barberet P, Pouthier T, Ritter S, Fischer B, Voss KO, et al.
No evidence for DNA and early cytogenetic damage in bystander cells after heavy-ion microirradiation at two facilities. Radiat Res 2009;171:530-40.
Crescenzi E, Palumbo G, de Boer J, Brady HJ. Ataxia telangiectasia mutated and p21CIP1 modulate cell survival of drug-induced senescent tumor cells: Implications for chemotherapy. Clin Cancer Res 2008;14:1877-87.
Jaroszeski M, Heller R, Gilbert R. Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery: Electrically Mediated Delivery of Molecules to Cells. Ch. 9. Totowa, NJ: Humana Press; 2000. p. 173-86.
Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al
. Electrochemotherapy - An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of European standard operating procedures of electrochemotherapy study. Eur J Cancer Suppl 2006;4:3-13.
Mir LM, Moller PH, André F, Gehl J. Electric pulse-mediated gene delivery to various animal tissues. Adv Genet 2005;54:83-114.
Neumann E, Kakorin S, Toensing K. Principles of membrane electroporation and transport of macromolecules. Methods Mol Med 2000;37:1-35.
Benov LC, Antonov PA, Ribarov SR. Oxidative damage of the membrane lipids after electroporation. Gen Physiol Biophys 1994;13:85-97.
Jordan ET, Collins M, Terefe J, Ugozzoli L, Rubio T. Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J Biomol Tech 2008;19:328-34.
Meaking WS, Edgerton J, Wharton CW, Meldrum RA. Electroporation-induced damage in mammalian cell DNA. Biochim Biophys Acta 1995;1264:357-62.
Moscone D, Miscini M. Determination of superoxide dismutase activity with an electrochemical oxygen probe. Anal Chim Acta 1988;211:195-204.
Bray RC, Cockle SA, Fielden EM, Roberts PB, Rotilio G, Calabrese L. Reduction and inactivation of superoxide dismutase by hydrogen peroxide. Biochem J 1974;139:43-8.
Dahle J, Angell-Petersen E, Steen HB, Moan J. Bystander effects in cell death induced by photodynamic treatment UVA radiation and inhibitors of ATP synthesis. Photochem Photobiol 2001;73:378-87.
Rugo RE, Almeida KH, Hendricks CA, Jonnalagadda VS, Engelward BP. A single acute exposure to a chemotherapeutic agent induces hyper-recombination in distantly descendant cells and in their neighbors. Oncogene 2005;24:5016-25.
Demidem A, Morvan D, Madelmont JC. Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells: A relay for chemotherapy? Int J Cancer 2006;119:992-1004.
Alexandre J, Hu Y, Lu W, Pelicano H, Huang P. Novel action of paclitaxel against cancer cells: Bystander effect mediated by reactive oxygen species. Cancer Res 2007;67:3512-7.
Hei TK. Cyclooxygenase-2 as a signaling molecule in radiation-induced bystander effect. Mol Carcinog 2006;45:455-60.
Börsch-Haubold AG, Pasquet S, Watson SP. Direct inhibition of cyclooxygenase-1 and-2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase. J Biol Chem 1998;273:28766-72.
Gutkind JS. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 1998;273:1839-42.
Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, et al.
Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 1994;265:966-70.
Wilkinson MG, Millar JB. Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J 2000;14:2147-57.
Shields JM, Pruitt K, McFall A, Shaub A, Der CJ. Understanding Ras: ′It ain′t over ′til it′s over′. Trends Cell Biol 2000;10:147-54.
McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW, Chang F, et al.
Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta 2007;1773:1263-84.
Liang X, So YH, Cui J, Ma K, Xu X, Zhao Y, et al.
The low-dose ionizing radiation stimulates cell proliferation via activation of the MAPK ERK pathway in rat cultured mesenchymal stem cells. J Radiat Res 2011;52:380-6.
She QB, Chen N, Dong Z. ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation. J Biol Chem 2000;275:20444-9.
Denchi EL. Give me a break: How telomeres suppress the DNA damage response. DNA Repair (Amst) 2009;8:1118-26.
Nuta O, Darroudi F, Trott KR. The Role of the telomere/telomerase system in the bystander effect. Radioprotection 2008;43:5.
Harley CB. Telomerase and cancer therapeutics. Nat Rev Cancer 2008;8:167-79.
Kulkarni A, Zschenker O, Reynolds G, Miller D, Murnane JP. Effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to DNA double-strand breaks in a human tumor cell line. Mol Cell Biol 2010;30:578-89.
Shiloh Y. ATM and related protein kinases: Safeguarding genome integrity. Nat Rev Cancer 2003;3:155-68.
Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003;300:1542-8.
McClintock B. The Stability of Broken Ends of Chromosomes in Zea Mays. Genetics 1941;26:234-82.
Sheridan J, Tosetto M, Gorman J, O′Donoghue D, Sheahan K, Hyland J, et al.
Effects of radiation on levels of DNA damage in normal non-adjacent mucosa from colorectal cancer cases. J Gastrointest Cancer 2013;44:41-5.
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