In vitro effects of radiation on human retinoblastoma cells

Retinoblastoma (Rb) is the most common intraocular tumor of childhood and has been demonstrated clinically to be very sensitive to external beam radiation (EBR). The purpose of this study was to determine the survival of Rb cell lines at different endpoints following irradiation. We also studied Rb-reconstituted cell lines postirradiation to gain insight into the role of Rb following DNA damage. Suspension cultures were exposed to single doses of 1, 2, 5, and 10 Gy. Following irradiation, cell viability and cell death was assessed for up to 72 hr using Trypan blue exclusion and acridine orange/ethidium bromide (AO/EB) staining, respectively. Morphological features were examined with light and electron microscopy (LM and EM). Clonogenic survival was assessed 2 weeks postirradiation. Cell cycle distribution was analyzed by flow cytometry. A time- and dose-dependent decrease in cell viability was induced in Y79 and WERI-Rb1 cells following irradiation, although not below approximately 45% for the times and doses used. A similar but much-reduced effect on viability was observed in Rb-reconstituted cells. AO/EB staining, LM, and EM revealed features characteristic of apoptosis following irradiation and a time- and dose-dependent increase in apoptosis was observed. For Y79 and WERI-Rb1 cells, 30-40% apoptosis was observed 72 hr after 10 Gy irradiation; however, apoptosis was much reduced in Rb-reconstituted cells (approximately 8% Y79LxRb28; approximately 18% WERILxRb8). Rb cell lines were extremely sensitive to radiation, although less so in Rb-reconstituted lines, with clonogenic survivals after 2 Gy (SF2) of 0.14 for Y79, 0.06 for WERI-Rb1, 0.36 for Y79LxRb28, and 0.19 for WERILxRb8. Postirradiation, a sub-G1 population was observed, consistent with radiation-induced apoptosis, and Rb-reconstituted cells displayed a prolonged G2 phase. The clonogenic survival parameters of Rb cell lines are consistent with clinical observations, where extreme sensitivity to irradiation has been reported. Additionally, Rb protein protected cells from DNA damage and may also play a role in radiation-induced G2 delay. This in vitro approach provides a useful model for further radiobiological studies of Rb.