Enhancement of in vitro and in vivo tumor cell radiosensitivity by the DNA methylation inhibitor zebularine

Abstract

1222

Aberrant DNA hypermethylation is a frequent finding in tumor cells, which suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145 and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1 and 14-3-3 σ) was determined as a function of zebularine exposure using methylation specific PCR and reverse-transcription PCR. Zebularine resulted in DNA demethylation in a time dependent manner, with the maximum loss of methylation detected by 48h. To determine whether zebularine enhances radiosensitivity, cell lines were exposed to zebularine for 24 or 48 h before irradiation and performance of the colony-forming efficiency assay. Exposure to zebularine for 24 h before irradiation resulted in an increase in radiosensitivity of each cell line with DEFs (dose enhancement factors at a surviving fraction of 0.1) of 1.25, 1.15 and 1.24 for MiaPaCa, DU145 and U251, respectively. Zebularine treatment alone resulted in surviving fractions of 0.93 ± 0.095, 0.66 ± 0.093 and 0.65 ± 0.068 for MiaPaCa, DU145 and U251, respectively. When the cell lines were exposed to zebularine for 48 h before irradiation there was a greater degree of enhancement of radiation-induced cell killing with DEFs of 1.51, 1.67 and 1.59 for MiaPaCa, DU145 and U251, respectively. Using this protocol the surviving fractions for zebularine exposure alone were 0.35 ± 0.066, 0.22 ± 0.038 and 0.42 ± 0.041 for MiaPaCa, DU145 and U251, respectively. As a measure of radiation-induced DNA damage, γH2AX expression was determined. Whereas zebularine had no effect on radiation-induced γH2AX foci at 1 h, the number of γH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 h after irradiation suggesting the presence of unrepaired DNA damage. These in vitro studies were then extended to an in vivo xenografts model. Zebularine administration to mice reactivated gene expression in U251 xenografts; irradiation of U251 tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay. These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.