Penetration of mouse fibroblasts by the 5'-phosphate of 9-beta-D-arabinofuranosyladenine and incorporation of the nucleotide into DNA

Abstract

9-beta-D-Arabinofuranosyladenine 5'-monophosphate (araAMP) is more lethal to mouse fibroblasts (L cells) than the identical exogenous concentration of 9-beta-D-arabinofuranosyladenine (araA) (Cancer Res. 32, 1512, 1972). [(3)H,(32)P]AraAMP (0.1 mM) was taken into L cells for 4 hr in the presence of a large excess of (32)P(i). The radioactivity was subsequently found mainly in the adenine nucleotides in the acid-soluble fraction and in the cell DNA. The cellular concentration of 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) exceeded 2 muM. More than 90% of the (3)H was associated with araA in the nucleotides. After degradation of the adenine-containing triphosphates with apyrase, the adenine mononucleotides were separated by thin-layer electrophoresis and chromatography. All of the (32)P and 97% of the (3)H were associated with araAMP. The small amounts of (3)H and (32)P in the acid-insoluble material were similar during a 4 hr incubation. The DNA fraction was degraded enzymatically to 5'-mononucleotides. Both (32)P and (3)H were associated predominantly with 5'-dAMP. Most of the (3)H was in araA, detected after dephosphorylation. Enzymatic degradation of the DNA fraction to 3'-mononucleotides and fractionation revealed (3)H primarily in the 3'-adenine nucleotide and (32)P in each of the deoxynucleoside 3'-monophosphates. After dephosphorylation of the 3'-mononucleotides, 93% of the (3)H was found in araA. These results suggest that small amounts of araAMP penetrated the cell as an intact nucleotide, were further phosphorylated to the triphosphate, and subsequently incorporated in internucleotide linkage into DNA.