Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors.
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.
Liu
M
M
Yale University School of Medicine, Department of Internal Medicine, New Haven, Connecticut 06520, USA.
Cao
D
D
Russell
R
R
Handschumacher
R E
RE
Pizzorno
G
G
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*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C034753:5-benzylacyclouridine; D014498:Uracil; D014543:Uridine Phosphorylase; D014529:Uridine