Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors

Cancer Res. 1998 Dec 1;58(23):5418-24.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Drug Screening Assays, Antitumor
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Humans
  • Kinetics
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Neoplasms / drug therapy
  • Neoplasms / enzymology*
  • Rabbits
  • Sequence Homology, Amino Acid
  • Uracil / analogs & derivatives*
  • Uracil / pharmacology
  • Uridine / metabolism*
  • Uridine Phosphorylase / biosynthesis
  • Uridine Phosphorylase / isolation & purification
  • Uridine Phosphorylase / metabolism*

Substances

  • DNA, Complementary
  • Enzyme Inhibitors
  • 5-benzylacyclouridine
  • Uracil
  • Uridine Phosphorylase
  • Uridine