Title: X-ray Microbeam Bystander
Studies With Human Mammary Epithelial Cells and Fibroblasts
Authors: E.A. Blakely1, R.I. Schwarz1, A.C. Thompson2,
K.A. Bjornstad1, P.Y. Chang1, 4, C.J. Rosen1, D. Sudar1,
R. Romano3 and B. Parvin3
Institutions: Divisions of 1Life Sciences,
2Advanced Light Source, and 3Computational Research, Lawrence
Berkeley National Laboratory, Berkeley, CA 94720-8174 USA
and 4SRI International, Menlo Park, CA 94025 USA.
The central
focus of our project is to use the unique synchrotron-based
source of a 12.5 keV X-ray Microbeam line 10.3.1 at the
Advanced Light Source (ALS) at Lawrence Berkeley National
Laboratory (LBNL) to quantitatively characterize low-dose
responses of low-LET, radiation-induced bystander effects
in a novel tissue-like model of human mammary epithelial
cells (HMEC), or normal human fibroblast cells (HFC), or
in a third scenario with both cells together in a co-culture
system.
In our experiments, cultures grown in microwell
slide chambers were irradiated with precise stripes of
dose up to 100µm
wide. Samples were processed for the expression of radiation-induced
protein markers with fluorescent immunohistochemistry in
a time course from 10 minutes to several hours after exposure.
Using fluorescence microscopy on a high-precision-controlled
microscope stage and fiducial marked references, the physical
locations of the dose stripes were mapped exactly to the
location of the biological responses. Computer-based fluorescent
analysis of radiation-induced signals in thousands of cells
has revealed statistically significant differences in the
broadening of the effects of the dose stripes to neighboring
unirradiated cells with time after exposure. Such broadening
of the dose stripe to involve cells not in the irradiated
field represents a radiation-induced bystander effect that
can be quantitatively evaluated. We demonstrated that the
sensitivity of detection in our model system is below 10
cGy, with dose stripes discernible after 5 cGy. The intensity
of the fluorescence was greater in the dose stripe for
larger doses (e.g. 100 cGy), and the fluorescence signal
decreased more slowly with time after high-dose exposure
than after lower doses (e.g. 25 cGy or 10 cGy). Results
from a rapid time course study show that radiation-induced
signals were observed within 10 min after exposure in cells
adjacent to, but outside of the irradiated area. The effect
was apparent at 10 min after exposure and diminished with
time, but was still significant 3 hrs after exposure. We
have quantitated dose-dependent induction of bystander
effects in several classes of radiation-induced signals
in our on-going studies and examined how radiation exposure
changes cell signaling acutely, and chronically.
Our complementary
screening of dose-dependent activation of gene expression
with high-throughput methods extends the number of candidate
genes that can be further studied. Using the Gene array
coupled with the quantitative RT-PCR validation approach,
we have obtained evidence demonstrating cell-type specificity
in the constitutive expression of genes, as well as dose-dependent
Xray-induced genes, known to be involved in ATM/ATR damage
responsive pathways. We have also demonstrated differences
in the constitutive, as well as low 10 cGy-induced, expression
of gap-junction connexin genes, suggesting cell-type
specificity in mechanisms of cell communication.
Our hypothesis
is that multicellular crosstalk following exposure to
low-dose or low-dose rates of low-LET ionizing radiations
may trigger signal transduction pathways that will deregulate
normal cell function in the irradiated, as well as neighboring
unirradiated cells, leading to bystander effects. These
studies in a relevant human cell model system may lead
to the development of low-dose radiation risk models
that include the effects of multicellular targets.
This work
was supported by the U. S. DOE’s
Low Dose Radiation Research Program under Contract No.
DE-AC03-76SF00098.
