Genomic Instability Induced by a Bystander Signal

Munira Kadhim
email: m.kadhim@har.mrc.ac.uk
Radiation and Genome Stability Unit, Medical Research Council, Harwell, UK.

Why this Project:

Radiation exposure can lead to a number of cellular changes that resemble those observed during tumorigenesis. Genomic instability describes an increased rate in the accummulation of new genetic alterations and is a hallmark of tumorigensis. 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). 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 extrapoliation of radiation exposure dosimetry and potential cancer risk associated with exposure to ionising radiation. Currently, the underlying mechanism for the induction and perpetuation of genomic instability, in both directly irradiated and bystander populations, is poorly understood.

Project Goals:

The purpose of this project is to investigate the link between initial and delayed events in irradiated and bystander cells. The damage response in irradiated and bystander cells will be characterized using genetically stable and unstable cells which have defects in DNA repair pathways.

Experimental Approach:

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 -H 2AX immunostaining and single cell gel electroporesis (comet) assay) and long term damage (using the chromosomal instability assay and the comet assay). Primary human fibroblast (HF-19), previously shown to be susceptible to the induction of instability, were plated. One hour later, polyester inserts were added containing HF-19 cells in 2mls of medium. Cells on the base were exposed to 0.5 Gy alpha particles 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 -H 2AX, 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.

Expected Outcome:

The number of γ -H 2AX foci increased in irradiated cells 30 and 90 minutes after the irridiation, but they did not significantly increase in the bystander population at the same timepoints. When cells were allowed to communicate for 30 minutes after irradation, damage was increased in irradiated cells, however 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.