THE UTILIZATION OF NUCLEOTIDES BY ANIMAL CELLS^*

Abstract

We have extended our studies on the toxicity of several arabinosyl nucleotides to L cells. In contrast to a short-term toxic effect of 9-beta-D-arabinofuranosyladenine (araA), both 9-beta-D-arabinofuranosyladenine 5'-phosphate (araAMP) and 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate produced sustained killing of the fibroblasts. 9-beta-D-Arabinofuranosyladenine 2',5'-cyclic phosphate and 9-beta-D-arabinofuranosyladenine-N1-oxide-5'-phosphate were not toxic, whereas 9-beta-D-arabinofuranosylhypoxanthine 5'-phosphate and adenosine 3',5'-cyclic phosphate had relatively slight effects on cell growth. 2',3'-Dideoxyadenosine 5'-phosphate was lethal to L cells, whereas 2',3'-dideoxyadenosine inhibited growth only slightly. The survival time of mice bearing Ehrlich ascites carcinoma was prolonged beyond controls equally well by treatment with either araAMP or araA. To explore the mechanism of nucleotide toxicity, we studied the metabolism of exogenous [3H, 32P]-araAMP. The double-labeled compound entered the cells very slowly and accumulated in both acid-soluble and acid-insoluble materials. 32P and 3H were associated with the adenine nucleotide fractions in ratios similar to that of the exogenous compound. After dephosphorylation of the acid-soluble fraction, greater than 85% of the 3H was associated with araA. Following degradation of the adenine-containing triphosphates with apyrase, all of the 32P and 97% of the 3H were associated with araAMP. Only small quantities of each label were associated with RNA and DNA. Following enzymatic degradation of DNA to 5'-monophosphates, most of both 3H and 32P were associated with the dAMP fraction in a ratio that was similar to that of the exogenous araAMP. After dephosphorylation of the 5'-monophosphates, greater than 90% of the 3H was contained in arabinosyl nucleosides. DNA was also enzymatically degreded to 3'-monophosphates. 3H was found primarily in the 3'-adenine nucleotide fraction with a slight amount in the nucleoside, whereas 32P was distributed among all the 3'-mononucleotides. After dephosphorylation of the 3'-monophosphates, 93% of the 3H was associated with araA. These results indicate that a small amount of araAMP penetrated the cell as the intact nucleotide, was phosphyorlated to the triphosphate and was subsequently incorporated within DNA.

Comparison of the effects of arabinosyladenine, arabinosylhypoxanthine, and arabinosyladenine 5'-monophosphate against herpes simplex virus, varicella-zoster virus, and cytomegalovirus with their effects on cellular deoxyribonucleic acid synthesis

“…When ara-AMP is given to humans, less ara-Hx may be formed. This may account for the finding by Cohen and Plunkett that cytotoxicity of ara-AMP is not reversible, whereas that of ara-A and ara-Hx is reversible (10). The apparent equivalence of ara-A and ara-AMP in the present studies may be due to the abundance of intracellular dephosphorylases in HFF tissue cultures.…”

Decomposition Pathways of the Mono- and Bis(Pivaloyloxymethyl) Esters of Azidothymidine 5′-Monophosphate in Cell Extract and in Tissue Culture Medium: An Application of the ‘on-line ISRP-Cleaning’ HPLC Technique

“…These phosphorylated derivatives are not active therapeutically in their own right because they do not cross lipid cell membranes efficiently (Leibman and Heidelberg, 1955;Roll et aI., 1956, Lichtenstein et al, 1960Cohen and Plunkett, 1975). Moreover, they are readily dephosphorylated on cell surfaces and in extracellular fluids by nonspecific phosphohydrolases (Schrecker, 1968;Ho, 1971).…”

Haloalkyl Phosphate Derivatives of AZT as Inhibitors of HIV: Studies in the Phosphate Region

“…In general the therapeutic properties of preformed nucleotides are no better than those of their nucleoside precursors; this being attributed to poor membrane penetration by the charged phosphates (Cohen and Plunkett, 1975). Many attempts have been made to prepare uncharged, masked phosphates as membrane-soluble delivery forms of the free bio-active nucleotides.…”