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Activator Protein Accelerates Dihydropyrimidine Dehydrogenase Gene Transcription in Cancer Cells

Departments of ¹ Translational Cancer Research and ² Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; and ³ Cancer Research Laboratory, Hanno Research Center, Taiho Pharmaceutical Co., Ltd., Saitama, Japan

Requests for reprints: Keiji Tanimoto, Department of Translational Cancer Research, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan. Phone: 81-82-257-5841; Fax: 81-82-256-7105; E-mail: ktanimo{at}hiroshima-u.ac.jp.

Dihydropyrimidine dehydrogenase is the most extensively investigated predictive marker for individual response to 5-fluorouracil. Clinical responses to the anticancer agent, along with various reports, have clearly shown that dihydropyrimidine dehydrogenase activity is closely correlated to its mRNA levels, but the regulatory mechanisms of its expression have remained unclear. We attempted to clarify the mechanisms and found that activator protein (AP-1) is probably one of the key factors in the transcriptional regulation of DPYD in cancer cells, and that phorbol 12-myristate 13-acetate (PMA) plus ionomycin treatment enhances transcription of DPYD via AP-1 activation. In this study, we characterized our previously subcloned 5' region of human DPYD, an 3.0-kb fragment (accession no. AB162145). Luciferase reporter assay showed that the clone showed strong promoter activities in 293T and HSC42 cells, and comparative analysis using 5' deletion mutants suggested the existence of several positive and negative regulatory regions, including putative binding sites for AP-1, SP-1, and nuclear factor-B. PMA/ionomycin treatment increased the mRNA level of DPYD in HSC42 cells, and electrophoretic gel mobility shift assay showed that the complex on the putative AP-1 binding site was drastically induced by PMA/ionomycin treatment. The complexes formed were competed out by preincubation with the cold-consensus AP-1 binding site, and the DNA binding complex formed on the site contained c-Jun and c-Fos, which are components of AP-1 transcription factor. We further identified the functional AP-1 binding site (nucleotide positions from –290 to –280), whose nucleotide mutations abolished PMA/ionomycin-induced DPYD promoter activation.

Key Words: DPYD • 5-FU • Promoter • Transcription • AP-1