GENE EXPRESSION PROFILING OF HIGH LET RADIATION EXPOSURE

S.A. AMUNDSON, 1 K.T. DO, 2 L.VINIKOOR, 2 and A.J. FORNACE, Jr. 2

Columbia University, Center for Radiological Research, 630 W. 168th St., VC11-215, New York, NY 10032,
2
National Institutes of Health, National Cancer Institute, 37 Convent Dr., 37-6144, Bethesda, MD 20892.
1

On the molecular level, exposure of mammalian cells to ionizing radiation induces damage in multiple cellular
compartments, resulting in complex biological responses, many of which are mediated through alterations in gene
expression. Activation of these complex signaling pathways leads to the altered expression of many genes,
including many encoding growth factors, cytokines, oncogenes, and genes involved in cell cycle, apoptosis, DNA
repair, translation control, mRNA stability, oxidative stress responses, inflammation, and tissue injury. The majority
of gene expression profiling studies of ionizing radiation response, however, have considered only low LET x-ray or
-ray exposures, and while there was some initial speculation that the molecular responses to diverse genotoxic
insults might be relatively similar, it is now becoming well established that there are distinct gene expression
signatures corresponding to different classes of stress agents. The clustered damage and highly complex DNA
lesions induced by densely ionizing radiation differ from those induced by low LET irradiation, and appear more
difficult to repair. Such complex damage might well be expected to initiate signaling pathways and resultant gene
expression changes distinct from those responding to the less complex damage of low LET irradiation.
In a study comparing gene induction in the human myeloid ML-1 cell line by low doses of -rays and 0.43 MeV
neutrons produced at the RARAF facility we found differences in the magnitude of induction of previously
characterized radiation-response genes. Preliminary microarray experiments also suggested there may be qualitative
differences in the genes induced. Neutrons of 0.43 MeV were chosen for this study as this energy had been reported
to have the highest RBE for a number of endpoints. Since the biological effects of this energy appear most different
from those of low LET, it seemed reasonable that if distinct damage signaling pathways are triggered by high LET,
their resultant gene expression patterns might be most discernable in such a comparison.
The human B-lymphoblastoid TK6 cell line has also shown a robust gene-expression response to low LET radiation,
while NH32, a targeted p53 knock-out cell line derived from TK6, adds additional power to this model system by
enabling studies targeted at this key signaling pathway and modulator of carcinogenesis. In a much broader study,
we have exposed TK6 and NH32 cells to similarly lethal levels of 12 different agents. The gene expression profiles
revealed by microarray hybridization contain both broad similarities and specific patterns of early gene expression
changes four hours after exposure. For instance, a set of genes was identified that broadly discriminated between
agents primarily causing DNA damage (0.43 MeV neutron, -ray, UVB, methyl methanesulfonate (MMS),
adriamycin, camptothecin, cis-platin, arsenite and hydrogen peroxide) and non-genotoxic agents (heat shock,
osmotic shock and tetradecanoylphorbol 13-acetate (TPA) exposure). Different sets of genes were also identified
that could discriminate between specific agents within either the genotoxic or non-genotoxic groups of treatments,
with very strong signatures being identified for both TPA and arsenite treatments. Furthermore, the inclusion of
NH32 in these studies allowed detection of strong p53-dependent gene expression response signatures that varied
among agents, and were almost completely absent among non-DNA-damaging stresses.
While the response to -rays and neutrons showed broad similarity to other DNA-damaging agents, a small group of
genes appeared to distinguish ionizing radiation from other types of DNA damaging agents. A number of genes also
appeared to show differential expression following low LET -ray and high LET neutron exposure. Although there
are too few experiments in this present study to truly define a signature of high LET, the results support the
hypothesis that unique patterns of gene expression may occur in response to high LET exposure, as well as in
response to more diverse agents. Future experiments adding comparisons of heavy ion exposure to the framework
established here have the potential to add greatly to our understanding of the molecular responses to high LET
radiations characteristic of the unique space radiation environment, and may lead to improved risk assessment,
radiation protection, and the development of informative biomarkers.