Aberrant Methylation of DPYD Promoter, DPYD Expression, and Cellular Sensitivity to 5-Fluorouracil in Cancer Cells

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Abstract
Purpose: Dihydropyrimidine dehydrogenase (DPD), the initial rate-limiting enzyme in the degradation of 5-fluorouracil (5-FU), is known to be a principal factor in clinical responses to the anticancer agent 5-FU, and various reports have clearly demonstrated that DPD activity is closely correlated to mRNA levels. However, the regulatory mechanisms of DPD gene (DPYD) expression remain unclear. In this study, the regulatory mechanisms have been intensively studied. Experimental Design and Results: A subcloned 3.0-kb fragment of the 5′ region of DPYD contains a total of 60 CpG sites, suggesting that methylation status may affect the repression of DPYD. The clone showed various promoter activities that were largely correlated with mRNA levels in most cell lines, except HSC3 and HepG2. Bisulfite sequencing analysis revealed that various CpG sites around the transcription start site were abnormally methylated in cells with low DPYD expression: Reversal of hypermethylation by 5-azacytidine treatment significantly increased DPYD expression in HSC3 and HepG2 cells that showed strong promoter activity. In HepG2, in vitro methylation of the DPYD promoter directly decreased promoter activity, and 5-azacytidine treatment restored higher DPYD expression in a dose- and time-dependent manner, along with decreased sensitivity to 5-FU. Conclusions: We found that DPD activity was controlled, at least in part, at the transcription level of DPYD and that aberrant methylation of the DPYD promoter region acted as one of the repressors of DPYD expression and affected sensitivity to 5-FU in cancer cells. Our new results could lead to a more precise understanding of the molecular basis of 5-FU response.

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