Background: Ionizing radiation (IR) is highly effective against colorectal cancer. The genes and the specific gene-products responsible for differential radiation sensitivity have not been entirely elucidated. We hypothesized, that due to cancer-specific alterations of the genomic methylation status, expression of gene products implicated in the radiation stress response may be responsible for the corresponding radiation sensitivity of different cell lines. Re-induction of methylation-silenced genes with methylation-modifying agents might revert radiation resistance. The aim of this study was to elucidate the effect of DNA-demethylating agent 5-aza-cytidine (AZA) in combination with IR on colorectal cancer in vitro. Methods: Four colorectal carcinoma-derived cell lines genetically distinct with respect to microsatellite instability, methylation status of p16, point mutations of p53, or K-ras, were investigated. The radiosensitizing effect of AZA was assessed in proliferation and clonogenic survival assays. The influence of various doses of AZA on the cell cycle function was assessed using two dimensional FACS analysis. The influence of additional combined treatment with AZA on the cell cycle function was defined. Changes of the methylation pattern after AZA exposure of target genes were investigated using methylation-sensitive dot blot assays and levels of methylation were determined after exposure to different dose levels. Results: Treatment with AZA in addition to IR enhanced the antiproliferative effect in all cell lines and two cell lines were strongly sensitized to IR after AZA. Clonogenic survival was reduced by IR combined with additional treatment with AZA in all but one cell line. FACS analysis revealed that AZA used at radiation sensitivity enhancing concentrations did not have any influences on the cell cycle distribution. Only at high cytotoxic doses of >1 mM of AZA, combined treatment with IR resulted in a enhanced apoptosis. However, the addition of AZA to IR treatment resulted in enhanced accumulation of cells in G2/M phase compared to G2/M arrest 24 h after IR alone. The cell cycle alterations caused by AZA diminished 48 and 72 h after treatment with IR. Radiosensitizing concentrations of AZA exerted demethylating effects on potential target genes, such as p16 and other genes, implicated in the stress response and radiation sensitivity assessed by methylation sensitive dot blot, however AZA-induced methylation were only partially revered after prolonged AZA withdrawal correlating with sustained radiation sensitivity. Conclusions: AZA-induced changes of the genomic methylation pattern enhance radiation sensitivity. RT-induced G2/M cell cycle modifications are more pronounced after pre-treatment with AZA. AZA represents a valid candidate agent for combined modality treatment with radiotherapy for colorectal carcinoma.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]