The allogeneic effect: bystander effect in the primary immune response in vitro.
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The bystander effect.
The bystander effect refers to the induction of biological effects in cells that are not directly traversed by a charged particle. The data available concerning the bystander effect fall into two quite separate categories, and it is not certain that the two groups of experiments are addressing the same phenomenon. First, there are experiments involving the transfer of medium from irradiated cells, which results in a biological effect in unirradiated cells. Second, there is the use of sophisticated single particle microbeams, which allow specific cells to be irradiated and biological effects studied in their neighbors; in this case communication is by gap junction. Medium transfer experiments have shown a bystander effect for cell lethality, chromosomal aberrations and cell cycle delay. The type of cell, epithelial vs. fibroblast, appears to be important. Experiments suggest that the effect is due to a molecule secreted by irradiated cells, which is capable of transferring damage to distant cells. Use of a single microbeam has allowed the demonstration of a bystander effect for chromosomal aberrations, cell lethality, mutation, and oncogenic transformation. When cells are in close contact, allowing gap junction communication, the bystander effect is a much larger magnitude than the phenomenon demonstrated in medium transfer experiments. A bystander effect has been demonstrated for both high- and low-LET radiations but it is usually larger for densely ionizing radiation such as alpha particles. Experiments have not yet been devised to demonstrate a comparable bystander effect on a three-dimensional normal tissue. Bystander studies imply that the target for the biological effects of radiation is larger than the cell and this could make a simple linear extrapolation of radiation risks from high to low doses of questionable validity.
Alpha Particles↗
Induction of stem cell cycling in mice increases their sensitivity to a chemical leukaemogen: implications for inherited genomic instability and the bystander effect.
Preconception paternal irradiation (PPI) modifies haemopoietic and stromal tissues of offspring and increases risk of generating lympho-haemopopietic malignancy if those offspring are then exposed to a leukaemogen. We hypothesised that this increased risk was related to inherited damage which had caused increased stem cell proliferation rates. To test for this link, in vivo, rapid stem cell proliferation was established by giving sub-lethal irradiation (3Gy gamma-rays) and allowing 3 days recovery. At this stage, 60% of haemopoietic spleen colony-forming units (CFU-S) were in DNA-synthesis, compared to <10% in unirradiated controls. Two groups of mice, unirradiated controls and irradiated animals, were then injected with 50mg/kg methyl nitrosourea (MNU) and observed daily for onset of lympho-haemopoietic malignancy. In a further control group of 60 mice, irradiated but not injected with MNU, only one leukaemia developed. In unirradiated controls, 20% of the mice developed malignancies between 3 and 8 months later: in the irradiated, MNU-treated groups, 95% developed malignancies between 2 and 7 months later. Thus, at least one powerful potentiating mechanism for induction of lympho-haemopoietc malignancy following inherited damage can be related to haemopoietic stem cell proliferation. Genomic instability is exposed by cell proliferation and has been implicated in this type of damage. However, a regulatory stromal microenvironment plays a part in inducing that proliferation. Thus, the microenvironment is the effective "bystander" which is thought to promote and amplify genomic instability, and thereby influence the induction of malignancy both in PPI offspring and in mice with induced stem cell proliferation.
Alkylating Agents↗
In vivo validation of the bystander effect.
The bystander effect post radionuclide decay describes the biologic response(s) of cells not directly targeted by the radiation insult. Recently, we demonstrated that the specific irradiation of human tumor cells in vivo leads to a bystander effect in subcutaneously growing tumors. These in vivo findings 1) call for the re-evaluation of approaches currently used for estimating the risks to individuals/populations inadvertently exposed internally to radioactivity (e.g., alpha particles) as well as to patients undergoing routine diagnostic nuclear medical procedures, and 2) impact significantly the current dogma for assessing the therapeutic potential of internally administered radionuclides.
Animals↗
A natural products chemist looks at the bystander effect!
The bystander effect is a process whereby the medium in which cells are irradiated at very low levels is presumed to contain a "low molecular weight" entity that will induce cell death and/or genetic changes in fresh mammalian cells when they are incubated in the medium. Following discussions at a recent meeting in McMaster University, suggestions are made in this paper as to the possibility for the "entity" to be nitric oxide and recommendations are given as to methods that will permit a systematic (bio)chemical search to be performed in order to identify the "activator" and prove/disprove the hypothesis.
Animals↗
Radiotherapy and the potential exploitation of bystander effects.
Radiation-induced bystander effects are the subject of intense investigation in radiation protection. The effects predominate at low doses and have been discussed mainly in terms of the impact on low-dose risk assessment. Possible therapeutic implications have been alluded to, but not discussed in any detail. The purpose of this review was to consider bystander biology in areas of major importance or interest in radiotherapy. These include consideration of radiation-induced bystander effects during the cell cycle, under hypoxic conditions, when fractionated therapy modalities are used, or when combined radiochemotherapy is given. Also discussed are individual variations in toxicity of bystander factors and normal tissue "collateral" damage. The importance of considering the tumor in the context of the organ, and even the organism that supports it, is also discussed. Direct clinical radiotherapy studies that consider bystander effects are not in the public domain at the time of writing, but many in vitro studies are available that are relevant; some preliminary animal data have also been published. Because radiation-induced bystander effects appear to challenge many of the central assumptions that underlie radiotherapy practice, it is important to consider what unexplored treatment avenues might result from a consideration of these effects. The final part of this paper is devoted to this point.
Bystander Effect↗
The effect of melanin on the bystander effect in human keratinocytes.
The influence of melanin on radiation-induced bystander effects has been studied. Melanin is known to be a natural substance with proved radioprotective properties in different organisms and cell lines. It is non-toxic and is effective against acute and chronic irradiation. The lower the radiation dose, the higher the relative impact of melanin protection. In this study influence of melanin on human keratinocytes (HPV-G cells) has been studied using the colony-forming assay. We have shown that bystander donor medium from 0.5 Gy irradiated cells when transferred to unirradiated cells, caused almost the same effect as direct irradiation. Melanin increased the colony-forming ability of bystander recipient cells when it was added into culture medium before irradiation. The effect of melanin added after irradiation was to produce less protection in both the directly irradiated and bystander medium treated groups. The absorption spectrum of the filtered medium is identical to one of the intact culture medium showing that melanin was not present in filtered medium. Thus, it cannot protect recipient cells but reduces the amount of the bystander effect. It is concluded that melanin added before irradiation effectively decreased the radiation dose. The reduction of the impact of the bystander signal on recipient cells when melanin was added to the donor medium after harvest but before filtration, may mean that the bystander signal has a physical component as melanin can absorb all types of physical energy.
Cell Communication↗
Cyclooxygenase-2 as a signaling molecule in radiation-induced bystander effect.
Radiation-induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the final biological consequences of exposure to low doses of radiation. Evidence suggests that targeted cytoplasmic irradiation results in mutation in the nucleus of the "hit" cells and that cells, which are not directly hit by an alpha particle, whether nuclear or cytoplasm, but are in the vicinity of one that does get hit, contribute to the genotoxic response of the cell population. Although radiation-induced bystander effects have been well documented in a variety of biological systems, the mechanism is not known. Using the Columbia University charged particle beam in conjunction with a novel strip dish design, we showed recently that the cyclooxygenase-2 (COX-2) signaling cascade plays an essential role in the bystander process. Treatment of bystander cells with NS-398, which suppresses COX-2 activity, significantly reduced the bystander effect as well as the induction of the mitogen-activated protein kinase (MAPK) pathways. These results provided the first evidence that the COX-2-related pathway, which is essential in mediating cellular inflammatory responses, is the critical signaling link for the bystander phenomenon. A better understanding of the cellular and molecular mechanisms of the bystander phenomenon together with evidence of their occurrence in vivo will allow us to formulate a more accurate model in assessing the health effects of low doses of ionizing radiation.
Animals↗
Genetically modified CD34+ cells exert a cytotoxic bystander effect on human endothelial and cancer cells.
We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34+ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34+ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34+ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34+ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34+ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34+ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34+ cells and total cell killing. For example, when 50% of CD34+ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80% of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34+ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.
Animals↗
A model for radiation-induced bystander effects, with allowance for spatial position and the effects of cell turnover.
Bystander effects, whereby cells that are not directly exposed to ionizing radiation exhibit adverse biological effects, have been observed in a number of experimental systems. A novel stochastic model of the radiation-induced bystander effect is developed that takes account of spatial location, cell killing and repopulation. The ionizing radiation dose- and time-responses of this model are explored, and it is shown to exhibit pronounced downward curvature in the high dose-rate region, similar to that observed in many experimental systems, reviewed in the paper. It is also shown to predict the augmentation of effect after fractionated delivery of dose that has been observed in certain experimental systems. It is shown that the generally intractable solution of the full stochastic system can be considerably simplified by assumption of pairwise conditional dependence that varies exponentially over time.
Bystander Effect↗
In vivo analysis of the 'bystander effect': a cytokine cascade.
The "bystander effect" refers to the death of unmodified tumor cells when in contact with ganciclovir (GCV)-exposed, herpes simplex virus-thymidine kinase (HSV-TK)-modified tumor cells. Although the exact mechanism or mechanisms involved in mediating the bystander effect in vivo are unknown, our findings suggest that an intact host immune system is required for the phenomenon to occur. The present study was designed to establish the effect of HSV-TK-modified tumor cells and GCV on the tumor and its microenvironment in vivo. In sublethally irradiated and immunodeficient Balb/c mice, the bystander effect was observed to be diminished or abrogated. Histopathologic examination of the tumor mass from immunocompetent mice demonstrated centralized hemorrhagic tumor necrosis (38%) after inoculation of the HSV-TK-modified tumor cells and GCV in tumor-bearing mice compared with the control mice (5%), indicating that cytokines such as tumor necrosis factor-alpha (TNF-alpha) were being released locally. This hypothesis was underscored using reverse transcriptase polymerase chain reaction (RT-PCR), by the demonstration of cytokine mRNA expression in mice treated with HSV-TK-expressing tumors and GCV. Semiquantitative PCR analysis for TNF-alpha using PCR-MIMIC on tumor samples from mice treated on days 1 and 4 showed a two-fold increase in the level on mRNA expression. Also, immunohistochemical staining for TNF-alpha showed that mononuclear inflammatory cells infiltrating the tumor were its source. Finally, characterization of tumor-infiltrating lymphocytes (TIL) in experimental animals demonstrated a two- to three-fold increase in the number of macrophages and T cells compared with control animals. These results demonstrate that, in vivo, the bystander effect is mediated in part by an antitumor response through the release of cytokines. Further, the cytokine milieu and tumor microenvironment can be modulated following injection of HSV-TK cells and GCV to enhance the host immune response, which is of potential use in clinical trials.
Animals↗
Bystander effects: a concept in need of clarification.
An increasing body of evidence indicates that the response to genotoxic agents such as radiation or drugs is a group phenomenon, rather than the summed response of individual independent cells to injury. Thus, a complex contagion-like response may spread beyond the initial impact of an agent to enlarge its effect. This indirect effect, termed "Bystander Effect," is multifaceted and may play a significant role in the therapy of tumors and in carcinogenesis. A better understanding of this phenomenon is needed in order to modulate treatment protocols to therapeutic advantage and to provide more rational guidelines for the evaluation of environmental hazards.
Animals↗
Bystander effects, adaptive response and genomic instability induced by prenatal irradiation.
The developing human embryo and fetus undergo very radiosensitive stages during the prenatal development. It is likely that the induction of low dose related effects such as bystander effects, the adaptive response, and genomic instability would have profound effects on embryonic and fetal development. In this paper, I review what has been reported on the induction of these three phenomena in exposed embryos and fetuses. All three phenomena have been shown to occur in murine embryonic or fetal cells and structures, although the induction of an adaptive response (and also likely the induction of bystander effects) are limited in terms of when during development they can be induced and the dose or dose-rate used to treat animals in utero. In contrast, genomic instability can be induced throughout development, and the effects of radiation exposure on genome instability can be observed for long times after irradiation including through pre- and postnatal development and into the next generation of mice. There are clearly strain-specific differences in the induction of these phenomena and all three can lead to long-term detrimental effects. This is true for the adaptive response as well. While induction of an adaptive response can make fetuses more resistant to some gross developmental defects induced by a subsequent high dose challenge with ionizing radiation, the long-term effects of this low dose exposure are detrimental. The negative effects of all three phenomena reflect the complexity of fetal development, a process where even small changes in the timing of gene expression or suppression can have dramatic effects on the pattern of biological events and the subsequent development of the mammalian organism.
Animals↗
Evidence for 'bystander effects' in vivo.
The observation of bystander effects in vitro have raised some serious questions as to the appropriate target size for calculation radiation dose. This has implications on the risk from ionizing radiation since dose is often directly related to radiation risk. This paper demonstrates that bystander effects do occur in vivo. It demonstrates that at low dose rates the bystander effects and risk are limited to the organ where the radiation dose is delivered. On the other hand, exposure to high radiation dose rates produces clastogenic factors that are released into the blood. These factors have been demonstrated both in vitro and in vivo and may influence risk in organs not directly exposed to the radiation. Bystander effects suggest that organs respond as a unit and are not just a bag of individual cells acting independently. Dose and risk must consider this unit.
Alpha Particles↗
Regressions of established breast carcinoma xenografts by carboxypeptidase G2 suicide gene therapy and the prodrug CMDA are due to a bystander effect.
The role of the bystander effect in the treatment of a human breast carcinoma xenograft was studied by suicide gene therapy with carboxypeptidase G2 (CPG2) and CMDA. Cells expressing enzymatically active surface-tethered bacterial CPG2 [stCPG2(Q)3] were mixed with control beta-galactosidase (beta-Gal)-expressing cells to give stCPG2(Q)3:beta-Gal ratios of, respectively: group 1, 0:100; group 2, 10:90; group 3, 50:50; and group 4, 100:0. Four days after injection of the cells into nude mice, the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) was administered. Tumor growth delay correlated well with the levels of stCPG2(Q)3 expression: group 1, 0 day delay; group 2, 10 days; group 3, 16 days; and group 4, 90 days. Similarly, the number of cures was strongly correlated to the levels of stCPG2(Q)3 activity: group 1, zero of six cured; group 2, one of six cured; group 3, three of six cured and group 4, four of six cured. There was a good correlation between CPG2 enzyme activity in the tumors and the number of cures. The majority of cells from groups 2 and 3 were apoptotic whereas those from group 1 were not, indicating a substantial bystander effect in the tumors. These results suggest that a bystander effect plays a major role in suicide gene therapy regimens with stCPG2(Q)3 and CMDA.
Animals↗
Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells.
A bystander effect is described when nontransduced or genetically unmodified cells are killed during death of genetically modified tumor cells transduced with a suicide gene. The "bystander effect" greatly enhances the efficacy of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy approach for cancer. The mechanism of the bystander effect is controversial. In this study, we examined the role of intercellular gap junction communication (GJIC) for the bystander effect in human gastrointestinal tumor cells. Our results show that the extent of the bystander effect varied amongst the tumor cell lines; pancreatic cancer cells BXPC-3 exhibited excellent bystander effects in vitro and in vivo studies whereas other gastrointestinal tumor cell lines such as pancreatic cancer cells MIAPACA-2, and colon cancer cells HT-29 showed poor bystander effects. Bystander effects were only found in the presence of cell-to-cell contact. The extent of the bystander effect was independent of the level of HSV-TK activity in the transduced tumor cells and was correlated with GJIC as demonstrated by an in vitro dye-transfer assay. Expression of the mRNA levels of gap junction protein connexin 43 was 8- to 26-fold or greater and connexin 26 gene expression was 2- to 229-fold greater in BXPC-3 cells compared to HT-29, MIAPACA-2, and PANC3 cells. Our results suggest that intercellular communication is essential for the bystander effect. The correlation between GJIC and the extent of the bystander effect suggest a role for GJIC in mediating the bystander effect. Analysis of tumors for GJIC or expression of gap junction proteins may identify the subset of patients suitable for gene therapy with the HSV-TK/GCV approach.
Animals↗
[In vitro and in vivo bystander effect of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene].
OBJECTIVE: To investigate in vitro and in vivo bystander effect, including distance bystander effect of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene (HSV-tk). METHODS: In vitro, mixed tk + BEL-7402 cells and tk-BEL-7402 cells in diverse proportions and ganciclovir (GCV) was given, then tested the survival ratio of cells by MTT. In vivo, 5 x 10(6) and 5 x 10(7) tk + BEL-7402 cells were injected into the tumors in nude mice following GCV. The change of the size of the tumors was observed. To observe distance bystander effect, adenovirues with HSV-tk (1 x 10(9) PFU) were injected into the tumor, which was the one of the bilateral tumors in nude mice. Subsequently, GCV was given and the change of the tumor was observed. RESULTS: Significant bystander effect was observed in vitro and in vivo. In vitro when tk + cells: tk(-) cells was 1:9, the survival rate of mixed cells was 36.6%. When the proportion of tk(+) cells was 90%, the survival rate of mixed cells was 3.2%. In vivo, those tumors with injection of tk(+) BEL-7402 cells were suppressed (P < 0.05). But in group of distance bystander effect the tumors on another side were not suppressed. CONCLUSION: In vitro, bystander effect exists. In nude mice, if tk(+) cells and tk(-) cells are contiguous, bystander effect is significant, or probably no bystander effect.
Adenoviridae↗
Potent bystander effect in suicide gene therapy using neural stem cells transduced with herpes simplex virus thymidine kinase gene.
OBJECTIVE: The herpes simplex virus thymidine kinase (HSVtk)/ganciclovir suicide gene therapy system has been considered as one of the most promising therapeutic strategies for malignant gliomas. We have been using HSVtk gene-transduced neural stem cells (NSCtk) that possess an ability to migrate toward a tumor mass for the treatment of experimental brain tumors. In the present study, we evaluated the potency of anti-tumor effect mediated by the bystander effect between NSCtk and C6 glioma cells in the HSVtk/ganciclovir suicide gene therapy system. METHODS: NSCtk and C6 glioma cells were mixed at various ratios (NSCtk:C6 cell ratios of 1:1 to 1:64) and the bystander effect was evaluated both under in vitro and in vivo conditions. RESULTS: In vitro co-culture experiment showed a complete tumor growth inhibition at the NSCtk:C6 ratios as low as 1:16. In vivo co-implantation study in the rat brain showed no visible tumors at the NSCtk:C6 ratios as low as 1:16 and all those rats survived more than 100 days. CONCLUSION: The results clearly demonstrated an extremely potent bystander effect between NSCtk and C6 cells, and the minimum number of NSCtk cells needed for the treatment of tumors was roughly estimated.
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