Enhanced Response of Radioresistant Carcinoma Cell Line to Heterogeneous Dose Distribution of Grid; The Role of High-Dose Bystander Effect.

PURPOSE: The classical dogma that restricted the radiation effect to the directly-irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully-exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect. Materials & Methods: Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using "medium transfer" and "cell-to-cell contact" strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly-exposed donors to 1.5 Gy uniformly-irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed.

RESULTS: Various parameters (ɑ/β, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 ± 0.06 in HeLa and 0.5 ± 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes ( Xrcc6 and H2afx ) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed.

CONCLUSIONS: Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.