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DOE Lowdose Radiation Program Workshop III

Abstract

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Title: Investigation of a Radiation-Induced Bystander Effect Using Co-culturing
Protocols.

Authors: Brian Ponnaiya, Gloria Jenkins-Baker, Mutian Zhang, Alan Bigelow, Stephen Marino and Charles R. Geard.

Previous studies in this laboratory using the charged particle microbeam have demonstrated the induction of micronuclei in fibroblast cells that were adjacent to alpha particle irradiated cells. To determine the suitability of the co-culturing approach to study the bystander effect, initial studies focused on the detection of the induction of micronuclei in irradiated and bystander fibroblasts.

Immortalized human fibroblasts (BJ1-htert) were irradiated with 0,0.1 (~1 per nucleus),1 and 10 Gy a–particles (90 keV/mm) and hit and bystander cells were examined for BrdU uptake and micronuclei incidence at 24, 48 and 72 hours post irradiation. As expected at 24 hours post irradiation there was a dose dependent reduction in the number of BrdU positive cells in the irradiated population (Figure 1). This was apparent at 48 and 72 hours as well. In addition, fibroblast bystander cells also demonstrated small though consistent delays in cell cycle progression.

Figure 1

Micronuclei frequencies observed in the irradiated and bystander populations are presented in Table 1. Yields of micronuclei in the irradiated populations were 2-4 fold higher than that of controls. The peak frequency was observed in the populations that received 1 Gy. The populations that received 10 Gy did not have significantly higher yields of micronuclei, which would be expected given the large delays in cell cycle progression observed. Bystander cells also had increased micronuclei frequencies at all doses. These increased yields were about 2 fold higher that controls at all doses, which is similar to the observations in the microbeam experiments. The similarities between these results and those of the microbeam experiments indicate that this is a suitable approach to study the bystander effect.

Table 1. Frequencies of micronuclei observed in co-cultured human fibroblasts and bystanders following a-irradiation.

Having detected the bystander effect in co-cultured fibroblasts, experiments were then conducted to determine whether a similar effect could be induced in co-cultured epithelial cells. Immortalized retinal epithelial cells (RPE-htert) were seeded onto mylar mylar rings and irradiated with 0,0.1,1 and 10 Gy a-particles (90 keV/mm). Epithelial cells seeded onto transwells served as bystander populations. Irradiated and bystander cells were examined for BrdU uptake and micronuclei incidence at 24, 48 and 72 hours post irradiation.

There was a dose dependent decrease in the number of cycling cells in the irradiated populations at all times points. Interestingly, unlike the bystander fibroblasts, the bystander epithelial cells did not appear to be delayed in progression through the cell cycle. Even at the earliest time point examined, 24 hours, there were no apparent differences between bystanders to irradiated populations and controls. There was a 2-4 fold increase in micronuclei yields in the irradiated populations, with the exception of the population that received the highest dose. Importantly, micronuclei frequencies in epithelial bystander populations were not increased as were those seen in fibroblast bystander populations. All of the epithelial bystanders had frequencies of micronuclei comparable to that seen in the controls. These data are at odds with microbeam results that indicated the induction of micronuclei in bystander epithelial cells.

Data presented here indicate that the co-culturing techniques developed in the laboratory are suitable for studying the bystander effect and may be useful in examining details of the phenomenon when used alone or in parallel with microbeam experiments. Studies are ongoing with low LET charged particles and photon sources to determine the relative significance of low dose low LET radiations in influencing bystander cell responsiveness.

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