The combination of 3 types of antipyrimidines was studied in AS-30D
hepatoma cells in
suspension culture and in the rat in vivo. Cellular
UTP and
CTP pools can be depleted most effectively by combining an inhibitor of de novo
UMP synthesis with
sugar analogs diverting
UMP to
UDP-
sugar analogs. The following
UMP-trapping
sugar analogs were employed: D-
galactosamine, D-
galactosone, D-
glucosone, and D-
glucosamine. These
D-galactose and
D-glucose analogs intensified the depletion of
UTP and
CTP pools induced by the following inhibitors of de novo
UMP synthesis:
acivicin,
PALA,
lapachol,
pyrazofurin, and
6-azauridine. The
sugar analogs, in the absence of inhibitors of the de novo pathway, enhanced the rate of de novo
UMP synthesis several-fold, as indicated by incorporation of 14CO2 into intermediates and products of the pathway and by the expansion of the
acid-soluble
uracil nucleotide pool. Reduction of
UTP and
CTP contents to less than 5 and 15% of control, respectively, by D-
galactosamine and
PALA resulted in a decrease of the rate of
RNA synthesis to 19% of control as calculated from the changes in specific activities of [14C]
CTP and of [14C]
cytidine in
RNA after labeling with [14C]
uridine.
Hepatoma cells released
uridine and
cytidine into the extracellular fluid. This release was reduced to one third in
UTP-deficient cells, indicating that
pyrimidine nucleoside excretion is regulated by
pyrimidine nucleotide levels, possibly by
UTP and
CTP regulation of
uridine kinase. Determination of the rates of de novo
pyrimidine synthesis, of the formation of
RNA pyrimidines, and of
pyrimidine nucleoside excretion indicates that de novo synthesis provides only about 67% of the
pyrimidines required for the consuming processes. The difference, as well as the dilution of labeled
pyrimidine nucleotide pools under conditions of a blocked de novo pathway, suggests a considerable salvage of
pyrimidine nucleosides derived from
RNA. This salvage of
pyrimidines may be intracellular and/or by an excretion and re-uptake process. Depletion of
UTP and
CTP pools, induced in
hepatoma cells by D-
galactosamine and
6-azauridine, leads to growth inhibition in
suspension culture; this inhibition becomes irreversible in an increasing percentage of cells, killing all cells after 20 hr of
UTP deficiency. The enhanced uptake of
5-fluorouridine by
UTP-deficient cells was associated with an increase of FUMP incorporation into
RNA up to 4-fold and with stronger inhibition of cell growth.(ABSTRACT TRUNCATED AT 400 WORDS)