12. Cytogenetic experiments failed to detect clear evidence for a negative bystander response in human lymphocytes cultured with irradiated cells

Abstract: 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.

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