title: [Expression of recombinant human dihydropyrimidine dehydrogenase and its application to the preparation of anti-DPD antibodies for immunochemical detection].
abstract: Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the catabolism of 5-fluorouracil (5-FU), and its expression in a tumor is thought to reduce the efficacy of 5-FU against the tumor. To detect a DPD molecule by immunoblotting and/or immunohistochemical methods, we attempted to prepare highly specific antibodies against recombinant human DPD (rhDPD) expressed in the baculovirus-expression system using hDPD cDNA. The expressed rhDPD protein was found to retain its entire molecular form and to show a high 5-FU-degrading activity equivalent to that of human liver DPD. Using this recombinant protein, both monoclonal and polyclonal antibodies to ehDPD were generated and their specificities, relationship to enzyme activity and the possibility of immunohistochemical measurement of tumoral DPD expression were investigated. The results revealed that anti-rhDPD monoclonal antibodies recognized only human DPD, while anti-rhDPD polyclonal antibodies reacted with both human and rodent DPD. The DPD content in 26 tumor cells, estimated by immunoblotting, was closely related to the 5-FU-degrading activities in those cells (r = 0.874). Moreover, immunohistochemical evaluation of tumor cellular DPD expression using our anti-rhDPD antibodies revealed that tumor cells expressing high levels of DPD showed strongly positive staining, but not those expressing low level or no DPD. These results suggest that immunochemical detection of tumoral DPD expression using our anti-rhDPD antibodies may be a means to predict the clinical response to 5-FU-based chemotherapy.
authors.affiliation: Second Cancer Laboratory, Taiho Pharmaceutical Co., Ltd.
authors.forename: H
authors.initials: H
authors.lastname: Okabe
authors.forename: K
authors.initials: K
authors.lastname: Arakawa
authors.forename: T
authors.initials: T
authors.lastname: Takechi
authors.forename: M
authors.initials: M
authors.lastname: Fukushima
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D042943:Dihydrouracil Dehydrogenase (NADP); D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010088:Oxidoreductases / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)