[Establishment of enzyme-linked immunosorbent assay for quantification of orotate phosphoribosyltransferase in gastric carcinoma].
A number of enzymes have been shown to be involved in the process of activation and/or degradation of 5-fluorouracil, and they are potential candidates for predicting factors of chemosensitivity to 5-fluorouracil. Among them, orotate phosphoribosyltransferase (OPRT EC 2.4.2.10) is a key enzyme related to the first-step activation process of 5-fluorouracil and therefore it has been shown to be an important enzyme for the prediction of sensitivity to 5-fluorouracil and its related derivatives. We developed a new enzyme-linked immunosorbent assay (ELISA) system to accurately assess intratumoral activity of orotate phosphoribosyltransferase. A new sandwich ELISA system was established using anti-OPRT polyclonal antibodies obtained from the rabbit immunized with the recombinant human peptides of the OPRT molecule. OPRT levels were measured in 8 human cancer xenografts transplanted in nude mice and 58 gastric cancer tissues using both a newly established ELISA and a conventional enzyme assay using radiolabeled 5-fluorouracil as a substrate. OPRT levels in 8 human cancer xenografts measured by this ELISA were significantly correlated with the OPRT enzyme activities (r2=0.782). Furthermore, OPRT activities measured in 58 gastric cancer tissues by enzyme assay were significantly correlated with those measured by the newly-established ELISA (r2=0.664). The ELISA system developed for the measurement of OPRT required a minimal amount of carcinoma tissue samples, which could be an easy-of-use assay system to predict sensitivity to 5-fluorouracil in gastric carcinoma. These results suggest that this newly-developed sandwich ELISA system for the quantification of OPRT is technically simple, feasible, and may be a useful tool to predict sensitivity to fluoropyrimidine-based anticancer chemotherapy in patients with gastric carcinoma and other cancers.
Sakurai
Yoichi
Y
Dept. of Surgery, Fujita Health University School of Medicine.
Sakamoto
Kazuki
K
Sugimoto
Yoshikazu
Y
Yoshida
Ikuo
I
Masui
Toshihiko
T
Tonomura
Shuhei
S
Inaba
Kazuki
K
Shoji
Mitsutaka
M
Nakamura
Yasuko
Y
Uyama
Ichiro
I
Komori
Yoshiyuki
Y
Ochiai
Masahiro
M
Matsuura
Shiro
S
Tanaka
Hideyuki
H
Oka
Toshinori
T
Fukushima
Masakazu
M
D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005472:Fluorouracil / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009962:Orotate Phosphoribosyltransferase / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000473:pathology
D009962:Orotate Phosphoribosyltransferase; D005472:Fluorouracil