Radiation enhancement by gemcitabine-mediated cell cycle modulations.

The purpose of this study was to investigate the exact dose dependency and time dependency of the radiation-enhancing effect of gemcitabine (2',2'difluoro desoxycytidine [dFdC]) in in vitro experiments (HeLa cells: cancer of the uterine cervix, #4197 cells: oropharyngeal squamous cell carcinoma), and to correlate this effect with the underlying changes in cell cycle distribution. Cell viability was determined fluorometrically after exposure to dFdC (0-20.0 micro mol/l), irradiation (0-37.5 Gy), and both modalities. Combining both therapies, cells were exposed to dFdC (0-10.0 micro mol/l) for 24 hours before further treatment and irradiated (0-30 Gy) immediately afterwards with or without removal of dFdC. For cell cycle analysis by flow cytometry, cells were irradiated (0-40 Gy) or treated with dFdC (0.012-1.0 micro mol/l, 24-48 hours). Additionally, cells were exposed to dFdC (2.0 micro mol/l, 0-4 hours). Cell cycle kinetics were evaluated using bromodeoxyuridine (BrdU) (10 micro mol/l) S-phase labeling, given either 30 minutes before or in the last hour of dFdC treatment (2.0 micro mol/l, 0-6 hours). The fluorometric assay revealed that dFdC enhances radiation-induced cytotoxicity at marginally toxic or nontoxic concentrations (<37 nmol/l). Radiation resulted in the anticipated G2/M arrest already at 2 Gy. DFdC induced concentration and exposure time-dependent cell cycle changes that were better resolved using BrdU, demonstrating a pronounced S-phase arrest already at 12 nmol/l. BrdU-pulse labeling revealed that the cell cycle block occurred at the G1/S boundary. Our data reconfirm the already known radiation enhancement, the S-phase specific activities of dFdC, and the relevance of the synchronized progression of cells through the S-phase with regard to the radiosensitizing properties of low-dose dFdC. However, we could demonstrate that before progressing in the S-phase, cells were blocked and partially synchronized at the more radiosensitive G1/S boundary. Furthermore, cells progressing past the block might accumulate proapoptotic signals caused by both radiation and dFdC, which will also results in cell death.