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.