title: Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors.
abstract: Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.
authors.affiliation: Yale University School of Medicine, Department of Internal Medicine, New Haven, Connecticut 06520, USA.
authors.forename: M
authors.initials: M
authors.lastname: Liu
authors.forename: D
authors.initials: D
authors.lastname: Cao
authors.forename: R
authors.initials: R
authors.lastname: Russell
authors.forename: R E
authors.initials: RE
authors.lastname: Handschumacher
authors.forename: G
authors.initials: G
authors.lastname: Pizzorno
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism*; D014543:Uridine Phosphorylase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C034753:5-benzylacyclouridine; D014498:Uracil; D014543:Uridine Phosphorylase; D014529:Uridine