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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