Combination Interferon-α2a and 13-cis-Retinoic Acid Enhances Radiosensitization of Human Malignant Glioma Cells in Vitro

We investigated the individual and combined effects of cis -retinoic acid (CRA) and/or IFN-α (IFN) and/or radiation therapy (RT) against a human glioma cell line (American Type Culture Collection; U373MG) to evaluate the possible radiosensitization properties of these agents in vitro . Glioma cells were incubated for 24 h in 96-well plates (2 × 10 2 cells/well) in standard culture medium. Sets of U373 ( n = 12) were exposed to CRA (3 × 10 6 μ m ), IFN (25 units/ml), CRA plus IFN, or standard culture medium. After an additional 24 h of incubation, the U373 cells were subjected to increasing radiation doses (up to 16 Gy). Glioma cells were harvested 92 h after irradiation, and cell survival curves were determined from [ 3 H]thymidine incorporation data (over the last 24 h). The experiment was repeated for both the untreated control group and the combined CRA/IFN group. To verify the [ 3 H]thymidine assays, a clonogenic assay was also performed. Single cell suspensions of U373 cells were plated out in six-well plates ( n = 3). After chemical and RT treatment, colonies of 50 cells or more were counted, and cell survival curves were generated as fractions of nonirradiated controls. The amount of RT (in Gy) that would cause a 50% survival fraction (lethal dose 50 or LD 50 ) was calculated from the survival curves by regression analysis. The following LD 50 s were obtained: The results showed that for both the [ 3 H]thymidine incorporation assay and the clonogenic assay, the combination of IFN/CRA rendered U373 cells more susceptible to ionizing radiation than the untreated control or either single agent alone.