Noguchi Takuya, Tanimoto Keiji, Shimokuni Tatsushi, Ukon Kei, Tsujimoto Hiroaki, Fukushima Masakazu, Noguchi Tsuyoshi, Kawahara Katsunobu, Hiyama Keiko, Nishiyama Masahiko
Department of Translational Cancer Research, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
Clin Cancer Res. 2004 Oct 15;10(20):7100-7. doi: 10.1158/1078-0432.CCR-04-0337.
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.
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.
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.
二氢嘧啶脱氢酶（DPD）是5-氟尿嘧啶（5-FU）降解过程中的初始限速酶，已知其是抗癌药物5-FU临床反应的主要因素，并且各种报告已清楚表明DPD活性与mRNA水平密切相关。然而，DPD基因（DPYD）表达的调控机制仍不清楚。在本研究中，对调控机制进行了深入研究。
DPYD 5'区域亚克隆的3.0 kb片段共包含60个CpG位点，提示甲基化状态可能影响DPYD的抑制。该克隆在大多数细胞系中表现出与mRNA水平高度相关的各种启动子活性，但HSC3和HepG2细胞除外。亚硫酸氢盐测序分析显示，在DPYD低表达的细胞中，转录起始位点周围的各种CpG位点异常甲基化：用5-氮杂胞苷处理逆转高甲基化显著增加了具有强启动子活性的HSC3和HepG2细胞中的DPYD表达。在HepG2细胞中，DPYD启动子的体外甲基化直接降低了启动子活性，5-氮杂胞苷处理以剂量和时间依赖的方式恢复了较高的DPYD表达，同时降低了对5-FU的敏感性。
我们发现DPD活性至少部分在DPYD的转录水平受到控制，并且DPYD启动子区域的异常甲基化是DPYD表达的抑制因子之一，并影响癌细胞对5-FU的敏感性。我们的新结果可能会使人们对5-FU反应的分子基础有更精确的理解。
