11. A bystander effect was found in co-cultured malignant trophoblast Jeg3 cells after 5 Gy X-rays using activation of p53 as endpoint
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Abstract: To study bystander effects, co-culture protocols were applied to examine the development of G2-arrest and the activation of p53 in a number of cell lines that were extensively characterized in term of their ability to establish gap juctional intercellular communication. A detailed set of experiments carried out with bEnd3 cells did not demonstrate bystander responses when either p53 activation or G2 arrest were used as endpoints. Measurements were carried out at several time points up to 24h after irradiation and co-culture. Because bystander responses are more robust after exposure of cells to high LET radiation, we also tested neutron irradiation using the same cell line. Here again no evidence for bystander responses could be obtained despite the outstanding capability of these cells to develop intercellular communication. Because bystander effects are known to be cell line specific, we tested additional cell lines using the same experimental protocols. For these experiments, cell lines were selected that were previously shown using different experimental approaches to elicit clear bystander effects.
In this set of experiments, Jeg3, A549, AG1522, T98G, EA-Hy926 as well as the repair deficient cell line xrs-5 were tested. Bystander responses could not be demonstrated in any of the cell lines. Similar experiments with Jeg3 cells carried out by the group at the Department of Anatomy provided evidence for the development of bystander effects that did not depend on gap junctional intercellular communication. The differences observed between the two groups in Essen in the development of bystander effects are not understood at present. They may reflect slight differences in the physiological conditions of the cells employed in the experiments (different sera and possibly slight differences in manipulation). They can also derive from differences between the two sorting instruments used. More work will be required to establish the source of these differences. Although the majority of the results obtained in these investigations are negative in terms of eliciting a bystander response, we believe that they are important in the field, as they allow narrowing down the conditions under which their development occurs.
Our observations when contrasted with reports of robust bystander responses using the same cell lines using different experimental approaches suggest that their development in the co-culture experiments may be prevented by the required manipulations such as trypsinization. In addition, they suggest that only a small fraction of the population responds to such signals making thus detection in the whole population difficult. To address some of the above issues we designed experiments using inserts in cell culture vessel that allow co-culture of two populations under conditions that allow free exchange of macromolecultes while preventing the mixing of the cell populations. In these experiments we used micronucleus formation as an end point for bystander response in the non-irradiated component of the culture. The preliminary results obtained in the experiments conduced with this system did not provide evidence for bystander response.
We investigated the expression of Caspase 3, an effector caspase at the end of the apoptotic cascade, as a further endpoint marker for bystander effects. Western blot analysis of activated Caspase-3 in irradiated Jeg3 cells showed no expression of activated Caspase 3 24 hours after irradiation. We conclude that activated Caspase 3 could not be used as an endpoint marker in Jeg3 cells for analysing bystander effects after X-ray irradiation.
Subject Descriptors: Radiation biology
Subject Index Codes: Life Sciences
Collaboration Sought: Information exchange/Training
Sources of Support: CEC
Remarks: Result eTIP
ILIAKIS, George (Professor)
UNIVERSITY OF DUISBURG-ESSEN
Essen, Kreisfreie Stadt