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

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of ur idine to uracil. We purified the enzyme from the murine colon 26 tumor usin g a two-step procedure through 5-amino-benzylacyclouridine affinity chromat ography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. T he polyclonal antibody used to screen a human liver expression library allo wed the isolation of a 1.2-kb done that contained the entire open reading f rame of the human UPase, The UPase cDNA has been expressed as a fusion prot ein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demo nstrated that the recombinant UPase preferentially uses uridine, 5-fluorour acil, and uracil as substrates, although lower levels of activity were obse rved with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues,were assayed f or phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Ac tivity in normal tissues appeared to be low but very sensitive to BAU (simi lar to 90% inhibition at 10 mu M). Tumors had generally 2-3-fold greater ac tivity compared with adjacent normal tissues. In breast cancer specimens an d head-neck squamous carcinomas, however, uridine cleavage was only partial ly inhibited (40-60%) by 10 or 100 mu M BAU, The BAU-insensitive activity r equires phosphate and pH conditions similar to the normal enzyme, and the n ew phosphorolytic activity was independent from thymidine phosphorylase. Th e BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosp horylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.