Columbia University Medical Center
Center for Radiological Research

NIH Program Project on
Radiation Bystander Effects: Mechanism
PO1-CA 49062-23

Overall Program Narrative

The present Columbia University Center for Radiological Research program project grant entitled “Radiation Bystander Effects: Mechanism” is an ever evolving program currently in the 23rd year funding. The program project was first funded in 1988 and was entitled “Radiation Biology of Simulated Radon-daughter Alphas”. The program direction was stimulated by, on the one hand, the pervading national interest in radon at that time and on the other hand, the beginning of the development of the Columbia single-particle microbeam, which was uniquely capable of addressing some basic issues of the radon problem. Research performed under the umbrella of this grant figured prominently in the BEIR VI Report entitled, “Health Effects of Exposure to Radon” and other national and international policy document. Over the years, the research focus has shifted from radon based risk assessment to the characterization and mechanism of radiation induced bystander effects. In 2005 the program was successfully renewed to focus on the mechanism of the bystander effects using in vitro and 3D human tissue models. In 2010, the application was renewed to ascertain the role of cyclooxygenase-2 (COX2), RAD9 and gap junctional proteins in modulating non-targeted responses of ionizing radiation utilizing both in vivo and in vitro model systems (Figure 1). In the next competitive renewal of this program, the goal is determine the health relevance of the non-targeted/bystander response and its possible role in radiation induced normal tissue damage and secondary malignancy.  


Fig. 1. A)  Heatmap of gene expression levels in directly irradiated (IR) and bystander human fibroblasts (BY) across the time course of 0.5, 1, 4 and 24 h as measured by low-density qPCR array. B) Working model of signaling pathways in non-targeted response.

What is a bystander effect?

For over a century since the discovery of X-rays, it was accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are due mainly to damage to DNA in the nucleus of a cell. As such, generations of students in radiation biology have been taught that such heritable biological effects are the consequence of a direct radiation-cell nucleus interaction. Using a charged particle microbeam where a single cell and/or a subcellular compartment can be selectively irradiated with either one or multiple particles, there is evidence that cells that are NOT directly traversed by a charged particle, but are in close proximity to cells that are, or that have received signals from such cells, can participate in the damage process. The bystander effect has revolutionized our conceptual thinking on the relevant target of ionizing radiation-induced DNA damage in that extranuclear and extracellular events can contribute to signaling events leading to DNA damage in both the targeted and non-targeted cells. In the ensuing years, a variety of biological endpoints in both human and other mammalian cell lines have firmly established the presence of a bystander effect under either confluent or sparsely populated culture condition (Figure 2). Furthermore, there is increasing evidence that progeny of bystander cells, which are not directly exposed to radiation may express a high level of genomic instability characterized by an increase in gene mutations, microsatellite instability and chromosome aberration. Since genomic instability is considered a predisposition factor for carcinogenesis, it has been postulated that radiation induced non-targeted/ bystander effects may promote secondary cancer induction in radiotherapy patients.

Figure 2. Bystander Cells

Fig.2. Bystander cells received signal from directly hit cells through either gap junctions or via soluble mediators

Non-targeted/abscopal effectA plethora of in vivo studies using a range of endpoints have since been demonstrated in non-targeted, out of field tissues/organs in a variety of organisms. Using X-rays, there is evidence that partial lung irradiation in mice can induce DNA damage in the un-irradiated part of the lung. Under the auspice of this Program Project grant, the Project Leader and other investigators in this program have recently shown that irradiation of a small 1 cm by 1 cm area of the lower abdomen of mice with X-rays results in the induction of COX2, a characteristic inflammatory molecule, as well as mutations in out of field lung and mammary tissues (Figure 3). Using a cytogenetic marker to distinguish donor bone marrow cells, there is evidence that descendants of irradiated stem cells, but not irradiated recipient stroma, are able to induce genomic instability in the descendants of non-irradiated stem cells. In addition, the production of inflammatory cytokines by macrophages is crucial in maintaining the long-term effects of the initial radiation exposure.

Figure 3.

Fig. 3. Non-targeted effects in the induction of COX2  and mutagenesis in out of field lung tissue in the gpt delta mouse model.  

Non-targeted effect and radiation-induced secondary tumorsRadiation is an important modality in the treatment of many types of human cancers and ~ 66% of cancer patients are treated with radiation either as a single treatment modality or as a neoadjuvant to surgery or chemotherapy. Furthermore, treatment of childhood cancers has become increasingly successful with a current overall cure rate of ~70%. On the other hand, radiation is also a well known human carcinogen and therapy-induced secondary cancer has become a matter of concern among long term survivors, particularly, among pediatric patients. There is evidence that ~8% of patients who are treated with radiation for their primary tumor develop a radiation-induced secondary tumor. Furthermore, among children who are treated with radiation for a childhood tumor, the incidence of second malignancy ranges from 18.4% in Hodgkin’s lymphoma to 8.8% for soft tissue sarcoma. Among prostate cancer patients where radiation is often the only treatment modality used, the incidence of calculated secondary tumor (non-prostate) from intensity modulated radiotherapy (IMRT) averages 5% for a 18 MV therapy unit.  There is evidence that among pediatric patients who developed therapy-induced secondary tumors, ~ 10~15% of these tumors arise at least 10 cm from the irradiated zone.  Although the contribution of scattering dose, however small it might be, to the observed cancers that arise far away from the treatment field cannot be completely ruled out, there are well designed laboratory studies using either X-rays or carbon ions (have minimal scattered dose) to quantify the contribution of scattered dose in the out of field response.  Hence, while these clinical findings are suggestive, nonetheless, the data provide a compelling argument of the potential role of non-targeted effects /genomic instability in human cancer.

The overall theme of the Program Project

This progam project brings together and links 3 projects that all address the common goal of understanding the how and why of the bystander phenomenon. The central hypothesis of the overall program is that the bystander effect involves multiple pathways and that an initiating event in the hit cells and a subsequent downstream signaling step involving the arachidonic acid cascade in the bystander cells play an important role in mediating the process.

Project 1 will harness the power of microarray profiling and functional genomics in order to gain insight into the cascade of signaling events between cellular targets and between cells. This study will be extended to a 3D tissue model as well as to single cells.
Project 2
will follow up on the preliminary observations that reactive nitrogen species may be involved in the signaling process and that the COX-2 enzyme is consistently elevated in bystander cells.
Project 3 will examine the contribution of genomic instability as a precipitating event in the induction of the bystander effect.

In between the projects, we will examine the gene profiling of nuclear versus cytoplasmic irradiation and whether the latter can induce bystander response in a manner similar to nuclear traversals. These studies are entirely dependent on the technology of the Columbia microbeam, which makes it possible to aim a defined number of α-particles (including one) at either the nucleus or cytoplasm of a cell with a precision of a few microns. The unequivocal demonstration of the bystander effect represents a paradigm shift in radiation biology since generations of students had been taught that heritable effects required the direct deposition of radiant energy in DNA. It is now apparent that the target for heritable damage is not only larger than the DNA, but larger than the cell itself.

Hypotheses to be addressed in this program

  • The bystander effect involves multiple pathways, and an initiating event in the hit cells and a subsequent downstream signaling step involving the arachidonic acid cascade in the bystander cells play an important role in mediating the process.

  • Both reactive radical species and signaling pathways involving the COX-2 gene are mediators of the bystander signaling process.

  • Gene expression signatures will reflect the signal transduction pathways responding to extranuclear, extracellular signaling and that interruption of these gene pathways using functional analyses can mitigate the bystander effects.

  • The basic signaling network mediating bystander response in cell culture system is similar in 3D tissue microenvironment.

  • Cytoplasmic irradiation can result not only in bystander effect, but in delayed chromosomal effects as well, and finally,

  • The signaling molecule(s) and mechanism(s) that mediate the bystander effect can also induce genomic instability in mammalian cells.

The three projects will be further linked together by a Technical Core, which will provide specialized irradiation facilities, data analyses, state of the art gene expression profiling, as well as proteomics support. In addition, an Administrative Core will provide infrastructure and oversight with respect to meetings, budgetary matters, critiques of manuscripts before submissions, as well as review and make decisions related to scientific direction.  At the same time, the laboratory projects interact with one another because each goal of the Program Project is addressed by more than one laboratory project, using different biological systems but with similar endpoints

Major Achievements (2009-date)

  • Demonstrated that Rad9 can control the direct and bystander irradiation induction of expression of Cox2, a key player in the bystander response and is frequently linked to cancer progression. We also showed that this regulation occurs at the level of transcription, as Rad9 can bind the Cox2 promoter specifically at two separate sites and regulate Cox2 RNA levels. 

  • Using a double mylar ring system, we optimized a protocol to identify cells that undergo a robust bystander response, and have used such cells to analyze the role of RAD9 in the response to direct versus bystander irradiation.  We found that RAD9 knockdown sensitizes cells to the bystander response, and by using microarray gene expression strategies we have discovered important signaling proteins.

  • We found that RAD9 promotes radioresistance in part by regulating the stability of integrin beta 1 protein.

  • We found that RAD9 is essential for prostate cancer stem cell viability. 

  • Demonstrated the induction of mutation and expression of Cox2 in out of field lung and mammary tissue in mice exposed to a single 5 Gy dose of X-rays in a small 1 cm by 1cm area in the lower abdominal area of the animals.

  • Demonstrated that pretreatment of irradiated animals with the specific Cox2 inhibitor, Nimesulide abrogates the non-targeted/ out of field response.

  • Using serial back-crossing, we successfully generated Cox2 knock out mice in the gpt delta C57BL/6 background. These animals, together with mouse embryo fibroblasts generated from pregnant animals are significantly less responsive to the bystander signaling observed in wild type animals.

  • Demonstrated the role of NFκB-dependent gene expression in mediating radiation induced non-targeted response in human skin fibroblasts. Induction of genes encoding interleukin 8 (IL8), IL6, PTGS2/COX2, tumor necrosis factor (TNF) and IL33 in directly irradiated fibroblasts produced the cytokines and prostaglandin E2 (PGE2) with autocrine/paracrine functions. As a result, bystander cells also started expression and production of IL8, IL6, IL33 and COX2-generated PGE2 to further promote the signaling process.

  • Demonstrated that mitochondrial DNA depleted (ρ0) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NFκB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ+ HSF.

  • Demonstrated signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). Neuronal differentiation of NSC was substantially inhibited by the environmental stressor through suppression of the PI3K-AKT signaling pathway.

  • Cranial irradiation that is widely used for treatment of brain tumors may induce death of neural stem cells (NSC) and cause substantial cognitive deficits such as impaired learning and memory. We demonstrated that reactive oxygen species induced by ionizing radiation dramatically up-regulated Tumor Necrosis Factor Related Apoptosis-Related Ligand (TRAIL) to stimulate TRAIL-receptor 2-mediated caspase-3-driven apoptosis in NSC.

  • Demonstrated directly irradiated NSC can initiate a bystander response in non-targeted NSC through paracrine mediated death ligands involving TRAIL signaling. The non-targeted response, including apoptosis, can be partially suppressed by anti-TRAIL antibody added to the cell media.
  • Demonstrated radiation-induced non-targeted, out of field effects including induction of COX2 and 8-OHdG in athymic nude mice.

  • Demonstrated that gap junction communication amplifies the toxic effects of densely ionizing radiation within hours following exposure, which is directly relevant to cancer radiotherapy.

  • Showed that human cell responses to ionizing radiation are differentially affected by the expressed connexins. The results revealed a prominent role for junctional permeability in determining the nature of the radiation response.

  • Demonstrated that persistence of oxidative stress in progeny of irradiated and bystander cells greatly depend on radiation quality and dose, which is relevant to understanding long-term health risks of exposure to ionizing radiation.

  • Proved that the progeny of bystander cells from cell cultures exposed to ionizing radiation are at a greater risk of neoplastic transformation, which is relevant to risk of developing second cancers following therapeutic irradiation of the primary cancer.

  • Characterized the kinetics of induction and decay of DNA damage in bystander cells from cultures exposed to low fluences of energetic particles, and demonstrated that the spread of dose due to secondary radiations is unlikely to contribute to stressful effects in cells not targeted by primary energetic particles.

  • Demonstrated and discussed the interplay between adaptive responses and stressful bystander effects in modulating biological responses to low and high doses/fluences of ionizing radiation at the molecular, cellular, tissue, and organ levels.

  • Unraveled a novel role for the Translationally Controlled Tumor Protein (TCTP), which is regulated in irradiated and bystander cells, in DNA damage sensing and repair.

  • Showed that micro RNA responses are differentially regulated in normal human cells exposed to chronic or acute low doses of g rays, which is relevant to issues related to radiation protection.

  • Demonstrated the role of junctional communication in the spread of toxic effects induced by different types of ionizing radiations delivered from a microbeam.

  • Characterized the crosstalk between junctional communication and COX2 in mediating the expression of radiation-induced bystander effects.

Members of the Program Project at the progress meeting on April 23rd, 2013
                at New Jersey Medical School of Rutgers University

Members of the Program Project at the progress meeting on April 23rd, 2013