Office of Biological and Environmental Research

DOE Low Dose Radiation Program Workshop V

2005 Abstract

Title: Genomic Instability Induced by a Bystander Signal

Authors: Munira Kadhim, Sharon Smyth, Stephen Moore, James Kelly and Edwin Goodwin.

Institutions: Radiation and Genome Stability Unit, Medical Research Council, Harwell, United Kingdom. Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM.

Ionising radiation is a potent mutagen and carcinogen. Radiation exposure can lead to a number of cellular changes that resemble those observed during tumorigenesis. Genomic instability describes an increased rate in the accumulation of new genetic alterations and is a hallmark of tumorigenesis. Genomic instability is observed in a fraction of the progeny of cells surviving direct radiation exposure and also in the progeny of cells that were never irradiated but communicated with irradiated cells (bystander cells). Currently, the underlying mechanism for the induction and perpetuation of genomic instability, in both directly irradiated and bystander populations, is poorly understood. The purpose of the project is to investigate the link between initial and delayed events in irradiated and bystander cells. We plan to further characterise the damage response in irradiated and bystander cells using genetically stable and unstable cells which have defects in DNA repair pathways.

In order to gain a clearer understanding of the relationship between damage induction and long-term effects in both irradiated and bystander cells, we assessed initial damage by γ-H2AX immunostaining and single-cell gel electrophoresis (comet) assay, and long-term damage using the chromosomal instability assay and the comet assay. Primary human fibroblasts (HF-19), previously shown to be susceptible to the induction of instability, were plated on the mylar base of individual dishes at a density of approximately 2 x 10⁵ cells per dish in 2mls of complete medium. One hour later, polyester inserts were added containing 1.5 x 10⁵ HF-19 cells in 2mls of medium. Cells on the base of the hostaphans were exposed to 0.5-Gy alpha-particles (²³⁸Pu; 3.2 MeV; 121.6 KeV/µm LET) and allowed to communicate with the bystander cells in the inserts for 0 min, 30 min, 90 min, and 24 hours before being removed and analysed using 3 different endpoints (γ-H2AX, comet, and cytogenetic analysis) for the irradiated and bystander populations. To assess the induction of delayed events typifying the genomic instability phenotype, populations were cultured separately for 2, 8, and 10 population doublings, at which time comet analysis and cytogenetic analysis was performed.

The number of γ-H2AX foci increased in irradiated cells at 30 and 90 min post-irradiation, but did not significantly increase in the bystander population after communication with irradiated cells via medium at the same timepoints. When cells were allowed to communicate for 30 min post-irradiation and analyzed by the comet assay, damage was increased in irradiated cells, but damage was not induced when cells communicated for 0 or 90 minutes. A similar trend was observed when the progeny were examined at passage 10, suggesting that the initial damage response is a critical determinant of the levels of damage observed at later times. Analysis of chromosomal aberrations is currently ongoing and will be presented and discussed.

The delayed effects of irradiation, such as genomic instability, have been observed in both irradiated and bystander cells under environmentally relevant exposure conditions. As such, both are extremely relevant for the extrapolation of radiation exposure dosimetry and potential cancer risk associated with exposure to ionising radiation.

This research was supported by the DOE Office of Science (Biological and Environmental Research), U.S. Department of Energy, Grant No.05475-SOL-05.

» Return to top