Researchers
with the U.S. Department of Energy (DOE)’s Lawrence Berkeley National
Laboratory (Berkeley Lab), through a combination of time-lapse live
imaging and mathematical modeling of a special line of human breast
cells, have found evidence to suggest that for low dose levels of
ionizing radiation, cancer risks may not be directly proportional to
dose. This contradicts the standard model for predicting biological
damage from ionizing radiation—the linear-no-threshold hypothesis or
LNT—which holds that risk is directly proportional to dose at all levels
of irradiation.
“Our
data show that at lower doses of ionizing radiation, DNA repair
mechanisms work much better than at higher doses,” says Mina Bissell, a
world-renowned breast cancer researcher with Berkeley Lab’s Life
Sciences Division. “This non-linear DNA damage response casts doubt on
the general assumption that any amount of ionizing radiation is harmful
and additive.”
Bissell
was part of a study led by Sylvain Costes, a biophysicist also with
Berkeley Lab’s Life Sciences Division, in which DNA damage response to
low dose radiation was characterized simultaneously across both time and
dose levels. This was done by measuring the number of RIF, for
“radiation induced foci,” which are aggregations of proteins that repair
double strand breaks, meaning the DNA double helix is completely
severed.
“We
hypothesize that contrary to what has long been thought, double strand
breaks are not static entities but will rapidly cluster into preferred
regions of the nucleus we call DNA repair centers as radiation exposure
increases,” says Costes.
As
a result of this clustering, a single RIF may reflect a center where
multiple double strand breaks are rejoined. Such multiple repair
activity increases the risks of broken DNA strands being incorrectly
rejoined and that can lead to cancer.”
Costes and Bissell have published the results of their study in the Proceedings of the National Academy of Sciences
in a paper titled “Evidence for formation of DNA repair centers and
dose-response nonlinearity in human cells.” Also co-authoring the paper
were Teresa Neumaier, Joel Swenson, Christopher Pham, Aris Polyzos,
Alvin Lo, PoAn Yang, Jane Dyball, Aroumougame Asaithamby, David Chen and
Stefan Thalhammer.
The
authors believe their study to be the first to report the clustering of
DNA double strand breaks and the formation of DNA repair centers in
human cells. The movement of the double strand breaks across relatively
large distances of up to two microns led to more intensely active but
fewer RIF. For example, 15 RIF per gray (Gy) were observed after
exposure to two Gy of radiation, compared to approximately 64 RIF/Gy
after exposure to 0.1Gy. One Gy equals one joule of ionizing radiation
energy absorbed per kilogram of human tissue. A typical mammogram
exposes a patient to about 0.01Gy.
Corresponding author Costes says the DNA repair centers may be a logical product of evolution.
“Humans
evolved in an environment with very low levels of ionizing radiation,
which makes it unlikely that a cell would suffer more than one double
strand break at any given time,” he says. “A DNA repair center would
seem to be an optimal way to deal with such sparse damage. It is like
taking a broken car to a garage where all the equipment for repairs is
available rather than to a random location with limited resources.”
However,
when cells are exposed to ionizing radiation doses large enough to
cause multiple double strand breaks at once, DNA repair centers become
overwhelmed and the number of incorrect rejoinings of double strand
breaks increases.
“It
is the same as when dozens of broken cars are brought to the same
garage at once, the quality of repair is likely to suffer,” Costes says.
The
link between exposure to ionizing radiation and DNA damage that can
give rise to cancerous cells is well-established. However, the standards
for cancer risks have been based on data collected from survivors of
the atomic bomb blasts in Japan during World War II. The LNT model was
developed to extrapolate low dose cancer risk from high dose exposure
because changes in cancer incidence following low dose irradiation are
too small to be measurable. Extrapolation was done on a linear scale in
accordance with certain assumptions and the laws of physics.
“Assuming
that the human genome is a target of constant size, physics predicts
DNA damage response will be proportional to dose leading to a linear
scale,” Costes explains. “Epidemiological data from the survivors of the
atomic bombs was found to be in agreement with this hypothesis and
showed that cancer incidence increases with an increase in ionizing
radiation dose above 0.1 Gy. Below such dose, the picture is not clear.”
Berkeley Lab biophysicist Sylvain Costes is generating 3D time lapse of DNA repair centers in human cells to understand better how cancer may arise from DNA damage. (Photo by Roy Kaltschmidt, Berkeley Lab) |
Previous
studies failed to detect the clustering of double break strands and the
formation of DNA repair centers because they were based on single-time
or single-dose measurements of RIF at a discrete time after the initial
exposure to ionizing radiation. This yields a net number of RIF that
does not account for RIF that have not yet appeared or RIF that have
already made repairs and disappeared. The time-lapse imaging used by
Costes, Bissell and their co-authors showed that RIF formation continues
to occur well beyond the initial radiation exposure and after earlier
repair issues have been resolved. Time-lapse imaging also indicates that
double strand break clustering takes place before any RIF are formed.
“We
hypothesize that double strand break clustering occurs rapidly after
exposure to ionizing radiation and that RIF formation reflects the
repair machinery put in place around a single cluster of double strand
breaks,” Costes says. “Our results provide a more accurate model of RIF
dose response, and underscore fundamental concerns about static image
data analysis in the dynamic environment of the living cell.”
Previous
studies also mostly involved fibroblast cells whereas Costes, Bissell
and their colleagues examined epithelial cells, specifically an
immortalized human breast cell line known as MCF10A, which has a much
higher background of RIF than fibroblasts, even without ionizing
irradiation. To compensate for this higher background, Costes developed a
mathematical method that enables background to be corrected for on a
per- nucleus basis in unirradiated cells. Still the use of a special
line of immortalized breast cells is an issue that Costes and his
colleagues plan to address.
“We
are now looking at primary breast epithelial cells that have been
removed from healthy donors to determine if our results are repeated
beyond just a single cell line and under more realistic physiological
conditions,” Costes says. “We’d also like to know if our findings hold
true for fibroblasts as well as epithelial cells. Also, we’d like to
know if double strand break clustering is the result of a random
coalescence or if there is an active transport mechanism that moves
these double strand breaks towards pre-existing DNA repair centers.”
Working
in collaboration with Rafael Gomez-Sjoberg of Berkeley Lab’s
Engineering Division, Costes and his group are also developing a special
microfluidics lab-on-a-chip device that is integrated into an X-ray
microbeam. The goal is to provide a means by which cells can be kept in a
controlled microenvironment while being irradiated with multiple doses.
This microfluidic array will be used to characterize DNA damage
response in breast and blood cells collected from human donors.
“By
characterizing DNA damage response in cells from many different human
donors,” Costes says, “we should be able to determine the variation
across humans and gain a better understanding of how sensitivity to DNA
damage from ionizing radiation might vary from individual to
individual.”
This research was supported by the DOE Office of Science.