Evidence for acyloxymethyl esters of pyrimidine 5'-deoxyribonucleotides as extracellular sources of active 5'-deoxyribonucleotides in cultured cells

Abstract

Cells commonly resist growth inhibition by purine and pyrimidine bases and nucleosides by restricting intracellular formation of the corresponding 5'-mononucleotides. Nucleotide derivatives that can act as effective membrane-transport precursors of the poorly membrane-permeable nucleotides have not been identified so far. We studied the bis(pivaloyloxymethyl)ester (I) of FdUMP (5-fluoro-dUMP) and a cyclic phosphodiester (II) of FdUMP derived from 1,3-dihydroxyl-1-C-(pivaloyloxy-methyl)propane which are active in vivo against a 5-fluoro-2'-deoxyuridine (FUdR)-resistant mouse leukemia and are attacked by carboxylic esterases under physiological conditions to produce FdUMP by elimination of formaldehyde and acrolein respectively. The assay for intracellular FdUMP was the inhibition of DNA synthesis due to inhibition of TMP synthetase in cultured mouse LM(TK-) fibroblasts genetically devoid of thymidine kinase (TK) and thus unable to convert FUdR directly to FdUMP. At 10(-6)M, I, II, or FUdR inhibited DNA synthesis in 2 hr by 99, 80, and 35% respectively; at 10(-5)M. maximal inhibition was attained after less than 15, 30 and 90 min respectively. Inhibition of DNA synthesis in TK+ cells by 10(-5) M I, II, or FUdR was reversed completely by 10(-5)M thymidine (TdR) but unaffected by 10(-5)M UdR, confirming TMP synthetase as the locus of inhibition. At 10(-5)M, bis(pivaloyloxymethyl) esters of phenyl phosphate or a p-substituted benzylphosphonic acid did not inhibit significantly DNA synthesis in TK+ cells. From this finding, and from effects produced by V (see below), we conclude that pivalic acid and CH2O arising from I contribute little to its above inhibitory effects. In TK- cells in which DNA synthesis is prevented by blockade of TMP synthetase with aminopterin, the bis(pivaloyloxymethyl) ester (V) of TMP, at 0.9 x 10(-4) M, induced a 4-fold faster rate of DNA synthesis than did 10(-3)M TMP, whereas 10(-3) M TdR did not affect the rate. After 3 hr the rate with V was 80% that in the absence of aminopterin. In the above systems the nucleotide diesters I, II and V appear to be acting as effective extracellular sources of active intracellular FdUMP and TMP, in processes that involve loss of the two esterifying groups.