Although radiation-induced bystander effects have been demonstrated in a number of cell types, the studies have largely been performed using high linear energy transfer (LET) radiation, such as a-particles. The literature is contradictory on whether fibroblasts show bystander responses, especially after low LET radiation such as X-or c-rays and whether the same signal transmission pathways are involved. Herein, a novel transwell insert culture dish method is used to show that X-irradiation induces medium-mediated bystander effects in AGO1522 normal human fibroblasts. The frequency of micronuclei formation in unirradiated bystander cells increases from a background of about 6.5% to about 9-13% at all doses from 0.1 to 10 Gy to the irradiated cells. Induction of p21 Waf1 protein and foci of c-H 2 AX in bystander cells is also independent of dose to the irradiated cells above 0.1 Gy. In addition, levels of reactive oxygen species (ROS) were increased persistently in directly irradiated cells up to 60 h after irradiation and in bystander cells for 30 h. Adding Cu-Zn superoxide dismutase (SOD) and catalase to the medium decreases the formation of micronuclei and induction of p21 Waf1 and c-H 2 AX foci in bystander cells, suggesting oxidative metabolism plays a role in the signaling pathways in bystander cells. The results of clonogenic assay of bystander cells showed that survival of bystander cells decreases from 0 to 0.5 Gy, and then is independent of the dose to irradiated cells from 0.5 to 10 Gy. Unlike the response with p21 Waf1 expression, c-H 2 AX foci and micronuclei, adding SOD and catalase has no effect on the survival of bystander cells. The data suggest that irradiated cells release toxic factors other than ROS into the medium.

The radiosensitizing e ect of ca eine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in ca eine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is a ected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays signi®cantly diminished ca eine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, ca eine e ectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not su cient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced ca eine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous endjoining (NHEJ) is radiosensitized by ca eine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly re¯ects NHEJ, remains una ected by ca eine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that ca eine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not su cient to explain radiosensitization. Indeed, immortalized ®bro-blasts from AT patients show ca eine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that ca eine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR. Oncogene (2000) 19, 5788 ± 5800.

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