DOE Low Dose Radiation Research Program Workshop I Abstracts November 10-12, 1999, Washington, D.C.

5. Effect of Low-Dose Alpha Irradiation in Human Cells: The Role of Induced Genes and the Bystander Effect

John B. Little, Hatsumi Nagasawa, and Edouard Azzam
Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115
gbraga@hsph.harvard.edu

Summary: The objective of this research program is to examine changes in gene expression and the induction of genetic damage in populations of normal human cells exposed to very low doses of alpha-particles whereby only a small fraction of the cell nuclei are traversed by a particle track.

Abstract: It has been long thought that the important genetic effects of radiation in mammalian cells are the result of direct DNA damage. Thus when cell populations are exposed to very low fluences of alpha-particle radiation, biological effects would occur only in those cells whose nuclei were actually traversed by a track. Presumably, no effects would be expected in the non-traversed 'bystander' cells in the population. We reported earlier (Cancer Res. 52, 6394, 1992) that when Chinese hamster ovary (CHO) cell populations were exposed to mean doses where only 1% of the nuclei are directly irradiated, an increased frequency of sister- chromatid exchanges occurred in 30-50% of cells in the population. In the present project, we are focusing on changes in the expression of genes involved in either cell cycle regulation or DNA damage recognition and repair in bystander human cells. Our specific aims are: 1) determine the extent of G1-phase delays under bystander conditions, and correlate these with changes in gene expression; 2) by use of immunofluorescence techniques, examine the in situ patterns of the expression of regulated proteins, investigate the effects of gap-junction inhibitors on gene expression, and confirm the involvement of intercellular communication in the response to low fluences of alpha-particles through the use of wild-type or connexin43 knockout cells; 3) determine the contribution of reactive oxygen species to the modulation of gene expression; 4) determine the involvement of extra-cellular or membrane originating signaling pathways; and establish whether the inhibition of activated signaling pathways (ATM/p53/p21Waf1) results in lack of enhancement of genetic damage in low-fluence exposed cells. Depending upon the results of the above experiments, we will extend these studies to cell strains derived from individuals with certain genetic disorders and knockout mouse strains for genes implicated in DNA repair (e.g., KU70, KU86).

Investigations are being performed in normal human diploid fibroblasts grown under stringently controlled conditions. Protein expression patterns and induction of genetic damage are being examined primarily by in situ microscopic techniques. Work in progress indicates that the G1 checkpoint is induced in cell populations exposed to mean doses as low as 1 cGy, and is entirely mediated by p53/p21Waf1. We have confirmed the involvement of gap-junction mediated intercellular communication in the regulation of the p53/p21Waf1 pathway, the modulation of expression of cell growth proteins and the induction of DNA damage in non-traversed bystander cells. Preliminary results indicate that the frequency of specific gene mutations may also be elevated in bystander cells. Finally, our data support the involvement of reactive oxygen species in the cellular response to low mean doses of alpha-particles. Overall, our findings indicate that biological effects are not restricted to the response of individual cells to the DNA damage they receive and that cell populations respond as a whole to radiation exposure, with communication occurring among the cells. These results imply that the modeling of dose-response relationships based on the number of cells hit may not be a valid approach.