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In this study, we investigated the potential influence of p53 on ultraviolet (UV) signal generation and response of bystander cells to the UV signals generated by beta-irradiated cells. Five cell lines of various p53 status (HaCaT, mutated; SW48, wild-type; HT29, mutated; HCT116+/+, wild-type; HCT116-/-, null) were irradiated with beta particles from tritium. Signal generation (photon emission at 340 ± 5 nm) was quantified from irradiated cells using a photomultiplier tube. Bystander response (clonogenic survival) was assessed by placing reporter cell flasks directly superior to irradiated signal-emitting cells. All cell lines emitted significant quantities of UV after tritium exposure. The magnitudes of HaCaT and HT29 photon emission at 340 nm were similar to each other while they were significantly different from the stronger signals emitted from SW48, HCT116+/+ and HCT116-/- cells. In regard to the bystander responses, HaCaT, HCT116+/+ and SW48 cells demonstrated significant reductions in survival as a result of exposure to emission signals. HCT116-/- and HT29 cells did not exhibit any changes in survival and thus were considered to be lacking the mechanisms or functions required to elicit a response. The survival response was found not to correlate with the observed signal strength for all experimental permutations; this may be attributed to varying emission spectra from cell line to cell line or differences in response sensitivity. Overall, these results suggest that the UV-mediated bystander response is influenced by the p53 status of the cell line. Wild-type p53 cells (HCT116+/+ and SW48) demonstrated significant responses to UV signals whereas the p53-null cell line (HCT116-/-) lacked any response. The two mutated p53 cell lines exhibited contrasting responses, which may be explained by unique modulation of functions by different point mutations. The reduced response (cell death) exhibited by p53-mutated cells compared to p53 wild-type cells suggests a possible role of the assessed p53 mutations in radiation-induced cancer susceptibility and reduced efficacy of radiation-directed therapy. [ABSTRACT FROM AUTHOR]

The induction of "bystander effects" i.e. effects in cells which have not received an ionizing radiation track, is now accepted but the mechanisms are not completely clear. Bystander effects following high and low LET radiation exposure are accepted but mechanisms are still not understood. There is some evidence for a physical component to the signal. This paper tests the hypothesis that bioelectric or biomagnetic phenomena are involved. Human immortalized skin keratinocytes and primary explants of mouse bladder and fish skin, were exposed directly to ionizing radiation or treated in a variety of bystander protocols. Exposure of cells was conducted by shielding one group of flasks using lead, to reduce the dose below the threshold of 2mGy 60Cobalt gamma rays established for the bystander effect. The endpoint for the bystander effect in the reporter system used was reduction in cloning efficiency (RCE). The magnitude of the RCE was similar in shielded and unshielded flasks. When cells were placed in a Faraday cage the magnitude of the RCE was less but not eliminated. The results suggest that liquid media or cell-cell contact transmission of bystander factors may be only part of the bystander mechanism. Bioelectric or bio magnetic fields may have a role to play. To test this further, cells were placed in a Magnetic Resonance Imaging (MRI) machine for 10min using a typical head scan protocol. This treatment also induced a bystander response. Apart from the obvious clinical relevance, the MRI results further suggest that bystander effects may be produced by non-ionizing exposures. It is concluded that bioelectric or magnetic effects may be involved in producing bystander signaling cascades commonly seen following ionizing radiation exposure. [ABSTRACT FROM AUTHOR]

We report data in this paper suggesting that fish irradiated to 0.5 Gy total body dose can release factors into the water that signal other unexposed fish and cause induction of bystander effects expressed as increased cell death in a reporter system. Radiation-induced bystander effects, resulting in the appearance of radiation damage or induction of typical radiation responses in unirradiated cells and tissues are now an established consequence of exposure to low doses of ionizing radiation, however little work has been done in vivo or in species other than humans or mice. In these experiments rainbow trout were irradiated and then paired with unirradiated fish for two hours. Additionally, unirradiated fish were placed in water which had previously been used to hold irradiated fish for 2 h. Sham-irradiated fish and absolute control fish were also examined all using blind protocols. Following a two h incubation period, at these various exposure regimes, the fish were killed by a blow to the head and dissected. Five organs were removed from each fish and tissue explants were cultured using an established technique. After 2 days, the culture medium was harvested and used in a reporter assay to determine whether a bystander effect had been induced. The explants were cultured on in Clonetics growth medium for a further 14 days then fixed for assay of radiation response proteins. The responses varied according to the cell type in the original explants, with the gill and fin showing the most pronounced response. The results suggest that communication signals leading to a typical radiation response can be passed between fish and seem to involve secretion of a chemical messenger into the water. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the relationship between two well-established delayed effects of ionizing radiation, experiments were conducted to determine the induction and expression of lethal mutations (delayed reproductive death) and chromosomal instability with respect to dose and time in a human immortalized keratinocyte cell line. Methods: HPV-G cells were γ- or α-irradiated and maintained in culture for up to 72 population doublings. At intervals, measurements were made of cloning efficiency and the cells examined for apoptosis and cytogenetic aberrations. Results: The descendants of cells surviving 1 or 3 Gy γ-irradiation, but not 0.5 Gy γ-irradiation, exhibited a reduced colony-forming efficiency. The reduction persisted at a constant rate of 15–20% clonogenic cell loss per population doubling for up to 72 population doublings. Apoptosis was demonstrated in all colonies in the 1 and 3 Gy groups at 30 and 72 population doublings post-irradiation but not in the 0.5 Gy group. A significant persistent reduction in colony-forming ability (~80%) was demonstrated in the progeny of cells irradiated with 0.5 Gy α-particles. After 30 population doublings, the proportion of chromosomally aberrant cells was significantly greater than control values for all doses of both high- and low-LET radiations. The major cytogenetic aberrations (chromatid breaks, chromosome fragments and minutes) were consistent with the transmission of chromosomal instability. The expression of instability declined between 30 and 72 population doublings in the 0.5Gy and 3 Gy gamma-irradiation groups, but persisted up to 72 population doublings in the 1 Gy group. The expression of chromosomal instability was greater in the descendants of alpha-irradiated cells and showed little evidence of reduction with time. Conclusions: Unstable aberrations characteristic of radiation-induced chromosomal instability may commonly result in apoptosis and account for a component of the delayed reproductive death/lethal mutation phenotype in HPV-G cells. However, the absence of lethal mutations in the descendants of 0.5 Gy γ-irradiated cells indicates a low-LET threshold effect for this particular endpoint. Overall, and particularly at low doses, there is no direct correlation between the two endpoints, indicating the absence of a simple relationship between these manifestations of radiation-induced genomic instability. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of irradiating microcolonies of various sizes on their subsequent ability to form colonies. Materials and methods: The location of individual HPV-G cells in tissue culture flasks was recorded using computerized microscopy. This allowed the cells' positions to be continually revisited, which enabled manual sizing of individual microcolonies both at the time of irradiation (between 0-5Gy) and following an incubation period during which the microcolonies were assessed for cologenic survival. Results: The experiments indicate that when irradiated as microcolonies, cells have plating e ciencies (PE) that are lower than expected when compared with the prediction from the PE values of cells irradiated individually. Conclusion: These data support the conclusions of Mothersill and Seymour (1997b) which challenge the concept of independent survival of certain types of cells following irradiation. The results indicate that interactions between cells can occur even in small aggregates. [ABSTRACT FROM AUTHOR]

Abstract: The influence of melanin on radiation-induced bystander effects has been studied. Melanin is known to be a natural substance with proved radioprotective properties in different organisms and cell lines. It is non-toxic and is effective against acute and chronic irradiation. The lower the radiation dose, the higher the relative impact of melanin protection. In this study influence of melanin on human keratinocytes (HPV-G cells) has been studied using the colony-forming assay. We have shown that bystander donor medium from 0.5Gy irradiated cells when transferred to unirradiated cells, caused almost the same effect as direct irradiation. Melanin increased the colony-forming ability of bystander recipient cells when it was added into culture medium before irradiation. The effect of melanin added after irradiation was to produce less protection in both the directly irradiated and bystander medium treated groups. The absorption spectrum of the filtered medium is identical to one of the intact culture medium showing that melanin was not present in filtered medium. Thus, it cannot protect recipient cells but reduces the amount of the bystander effect. It is concluded that melanin added before irradiation effectively decreased the radiation dose. The reduction of the impact of the bystander signal on recipient cells when melanin was added to the donor medium after harvest but before filtration, may mean that the bystander signal has a physical component as melanin can absorb all types of physical energy. [Copyright &y& Elsevier]