Uridylate-trapping sugar analogs in combination with inhibitors of uridylate synthesis de novo and 5-fluorouridine.
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)
Keppler
D
D
Fauler
J
J
Gasser
T
T
Holstege
A
A
Leube
K
K
Schulz-Holstege
C
C
Weckbecker
G
G
D000818:Animals; D002478:Cells, Cultured; D004357:Drug Synergism; D005260:Female; D005690:Galactose / Q000031:analogs & derivatives*; D005947:Glucose / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D011743:Pyrimidines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D011742:Pyrimidine Nucleotides; D011743:Pyrimidines; C001943:5-fluorouridine; D012313:RNA; D014542:Uridine Monophosphate; D005947:Glucose; D014544:Uridine Triphosphate; D014529:Uridine; D005690:Galactose