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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.
Irradiated Go lymphocytes were fused to exponentially growing CHO cells and cultured for up to 30h. In the hybrid cells, lymphocyte PCCs were induced when CHO entered mitosis. Cells were harvested at various times after cell fusion using shake off and subsequently chromosome preparations were made following standard procedures. Bystander effects in the hybrid cells between the irradiated lymphocyte nucleus and the unirradiated CHO nucleus could not be observed. In another set of experiments, irradiated exponentially growing CHO cells were fused to non-irradiated Go lymphocytes and cultured for up to 30 h. In the hybrid cells, lymphocyte PCCs were induced when CHO entered mitosis. Cells were harvested at various times after fusion using shake off and chromosome preparations were made. Bystander effects between irradiated mitotic CHO cells and non-irradiated Go lymphocytes were examined.
At various harvest times, the chromosomal damage in CHO cells involved in cell fusion and formation of hybrid cells was striking less than that scored in non-fused CHO metaphase cells at 3 Gy. When the dose in the CHO cells was increased to 6 Gy a similar response was observed. It is evident that this interesting "protective" or "negative" bystander effect needed further investigation. To test whether the bystander effects occur only in cycling cells and not in Go cells, experiments were carried out using PHA-stimulated peripheral blood lymphocytes fused to exponentially growing CHO cells exposed to 3Gy gamma-rays. By harvesting hybrid cells using the shake-off method, chromosome preparations were made and G1 or G2 lymphocyte-PCCs were scored for chromosomal damage to detect bystander effects. The results could not confirm the presence of such effects. Using the G2-assay, we classified 20 healthy individuals according to their G2-chromosomal radiosensitivity. To study whether bystander effects preferentially occur in the G2-radiosensitive donors, three types of experiments were carried out.
Firstly, peripheral blood from male donors was irradiated with 2, 4 and 6 Gy gamma-rays and immediately mixed with blood from G2-radiosensitive female donors. The mixed cells were cultured in the presence of PHA, and chromosomal aberrations were scored in non-irradiated lymphocytes. No bystander effects could be detected with cells from any of the donors including the radiosensitive ones. Experiments with irradiated exponentially growing CHO cells fused to peripheral blood lymphocytes from radiosensitive donors, as well as experiments with irradiated exponentially growing CHO cells fused to PHA-stimulated peripheral blood lymphocytes from radiosensitive donors, were also carried out. The results obtained in G0, G1 or G2 lymphocyte PCCs did not show evidence for bystander phenomena.
In order to study the parameter of LET and cell type on the development of bystander effects using cell survival as endpoint, we carried out experiments with primary human skin fibroblasts (HSF2). Using 4.5MeV alpha particles (LET = 100keV/µm) we were unable to measure bystander effects. This suggests that bystander responses are species dependent and more pronounced in rodent cells. After exposure of HSF2 cells to 10MeV microbeam protons (LET = 4.7keV/µm, comparable to 100kV X-rays), a significantly higher survival (compared to unirradiated controls) was observed for doses lower than about 0.3 Gy, when all cells or when only 10% of all cells were irradiated.
These results indicate protective rather than negative bystander responses. Considering that the plating efficiency of HSF2 cells is about 20%, it is likely that low doses stimulate processes that increase plating efficiency and compensate the negative effects of radiation. Doses in the range from about 0.6 to 2 Gy given to 10% of all cells yielded survival levels ranging from 1.0 to 1.1, thus showing no (negative) bystander effects. In summary, the data suggest that LET, dose and cell species are important parameters for the development of bystander effects. Interesting is the observation that primary human fibroblasts which are competent to form gap junctions show no bystander effect after high LET exposure, an effect clearly seen with immortalised rodent fibroblasts (C3H10T1/2 cells). This observation suggests that immortalisation or cell, origin of the cell (i.e. human or rodent) etc. may play a significant role in the development of bystander effects.
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