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 17th 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 we thought was uniquely capable of addressing some basic issues of the radon problem. Over the years, the research focus has shifted from radon based risk assessment to the characterization and mechanism of radiation induced bystander effects. 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.

What is a 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. There is evidence that targeted cytoplasmic irradiation results in mutation in the nucleus of the hit cells and that cells that are not directly hit by an alpha particle, whether nuclear or cytoplasm, but in the vicinity of one that does, contribute to the genotoxic response of the cell population. In this regards, the unique Columbia University charged particle microbeam (Technical Core) that can target either cellular nuclei or cytoplasm with a defined number of either protons or alpha particles with high precision, has played a pivotal role in the advance of the bystander field. The demonstration of a bystander effect in 3D human tissues and, more recently, in whole organisms have clear implication of the potential relevance of the non-targeted response to human health. The observations that the progeny of non-targeted cells show an increase in genomic instability as evidenced by an increase in delayed mutations and chromosomal aberrations many generations afterwards indicate the need for a comprehensive assessment of the bystander issue, particularly among genetically susceptible populations. Thus far, most of the published data on bystander effects have been largely phenomenological in nature. The overall theme of this program project is to define and characterize the mechanism of this non-targeted response using both in vitro and in vivo models. Mechanistic-based studies that can provide insight on the nature of the signaling molecule(s) will be invaluable in assessing the clinical relevance of the bystander effect and ways in which the bystander phenomenon can be manipulated to increase therapeutic gain in radiotherapy.

This program 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.

Major Achievements (2005-date)

  • Demonstrate the presence of a non-targeted, out of field mutagenic response in the lung tissue of irradiated gpt delta transgenic mice and that the lung tissues show a 20 fold increase in COX-2 protein levels 24 hours post-treatment (Chai et al., 2008; 2009).
  • We have shown that mouse embryo fibroblasts from Rad9 KO mice demonstrate a two fold increase in bystander apoptosis (Zhu et al., 2005).
  • Using the Columbia University microbeam, we have demonstrated a 3 fold increase in COX-2 among bystander human fibroblasts. Furthermore, treatment with NS398, a COX-2 inhibitor suppressed the COX-2 induction and obliterated the bystander mutagenesis (Zhou et al., 2005).
  • We have developed molecular interaction networks of the response to direct and bystander irradiation in primary human cells, and shown that while two primary hubs, p53 and NFκB, coordinate the response to direct irradiation, the bystander response is dominated by the NFκB hub and a network around β-catenin (Ghandhi et al. 2008).
  • We have investigated the time course of gene expression during the first 24 hours of bystander response, and identified a novel signaling axis involving IL33 and bystander activation of the AKT pathway in primary human cells as early as half an hour after treatment (Ghandhi et al. 2008).
  • We have demonstrated that the TNFα-NFκB-COX-2/PGE2 and the TNFα-NFκB-iNOS signaling pathways, which are hallmarks of inflammation, reactive oxygen and nitric oxide production, are critically linked to radiation bystander phenomenon in normal human fibroblasts (Zhou et al., 2008).
  • We have shown that the basal and inducible (both directly irradiated and bystander) levels of nuclear NF-κB DNA-binding activity were significantly higher in human skin fibroblasts compared to mitochondria-deficient ρ0 fibroblasts. Consequently, expression levels of NF-κB-dependent proteins such as iNOS and COX-2 were notably lower in ρ0 cells. Taken together, these results indicated that inducible (but not basal) expression of COX-2 and iNOS, which was substantially lower in mitochondria-deficient cells, plays a critical role in regulating mechanisms of bystander effects (Zhou et al., 2008)
  • Using a nematode Caenorhabditis elegans strain with a green fluorescent protein (GFP) reporter for the heat shock protein 4 (Hsp4), we have shown that targeted proton irradiation induces a non-targeted /bystander effect after 24 hr at a site as far as >150 µm away from the irradiated spot (Bertucci et al., 2009).
  • We established a model of interactions between radiation-induced oxidative stress, protein and DNA damage (Shuryak et al., 2009), and a biophysical model of radiation induced bystander effect (Shuryal et al., 2007).
  • Using the Columbia University microbeam we have provided a definitive demonstration of similar molecular responses in known hit versus known non-hit near by bystander cells by subjecting individual micro-manipulated cells to single cell RT-PCR (Ponnaiya et al., 2007).
  • We have consistently recorded enhanced genomic instability in the progeny of irradiated cells, of cells that were the progeny of non-hit bystanders to microbeam directed nuclear irradiated cells, and progeny of precise cytoplasmically irradiated cells (Hu et al., 2009).
  • We have evaluated the lateral extent of phosphorylation-protein induction for members of signaling pathways associated with COX-2 mediation of cellular responsiveness, and found significant induction within 1hr up to 1mm from the microbeam defined zone of irradiation in 3 dimensional model human skin (EpiDerm) samples.
  • We have evaluated DNA damage/repair responses in irradiated 3-D EpiDerm constructs compared with 2D human fibroblasts and found higher levels of DNA dsb repair protein foci in the latter. Pretreatment of the constructs with a PI 3 kinase like kinase (PIKK) inhibitor significantly diminished repair protein foci formation and increased apoptotic cell death, indicating a role for this signaling pathway (Su et al., 2009).
  • We have replaced the 55-year old 4.2-MV Van de Graaff with a new 5.5-MV Singletron accelerator from High Voltage Engineering Europa. This increases the energy stability of the charged particle beams and extends the available particle energies and ranges (Brenner et al., 2007).
  • Installed a compound electrostatic triplet quadrupole lens system and attained a sub-micron charged particle beam spot diameter (Randers-Pehrson et al., 2009).
  • Development of a “point-and-shoot” system for the microbeam to direct the beam to the target magnetically rather than move the target to the beam using the microbeam stage (Harken et al., 2008).
  • Development of a multiphoton microscope system for 3-D imaging using a laser with a wavelength range from 680 to 1080 nm for biological irradiation. (Bigelow et al., 2008).

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