Penetration of mouse fibroblasts by 2′‐deoxyadenosine 5′‐phosphate and incorporation of the nucleotide into DNA

Abstract

The metabolism of 2'-deoxyadenosine S'-phosphate (dAMP) by exponentially growing mouse fibroblasts (L-cells) in suspension culture has been studied. Cells incubated for four hours with 0.10 pmole [3H,32P]dAMP/ml in medium containing 9.6 pmole 31Pi/ml incorporate both activities at nearly linear rates into acid-soluble and acid-insoluble fractions. The 3H/32P value increased about 30-fold in each fraction during the incubation, indicating extensive dephosphorylation of dAMP. The DNA from treated cells was degraded enzymatically to 5'-mono-nucleotides, which were fractionated by ion-exchange chromatography. 3H was associated exclusively with dAMP (53%) and dGMP (47%). 32P, associated with all deoxynucleotides, was at a 20-fold higher specific activity in dAMP than in either dGMP or dCMP. The specific activity of [ 32P]dAMP incorporated into DNA in four hours was 24-fold greater than that of 32Pi in the cellular pool. In experiments in which cells were incubated with 32Pi plus or minus 0.10 @mole dAMP/ml, the specific activity of dAMP was slightly less than that of dGMP or dCMP, which were equal. These results suggest that the higher specific activity of [32P]dAMP in the DNA of cells after incubation with doubly-labeled dAMP was due to the intact penetration of some dAMP into the cells with its subsequent incorporation into DNA. Calculations, based on the amount of exogenously added [3H]thymidine incorporated into the cellular DNA in parallel cultures and the 32P of dAMP isolated from this DNA, suggest that 1% of the total dAMP residues incorporated during the 4-hour incubation were derived directly from intact, exogenously added dAMP.We have previously reported that upon incubation of L-cells with the toxic nucleotide 9-P-D-arabinosyladenine 5'-phosphate (araAMP), small quantities of the compound entered the cells without prior dephosphorylation (Plunkett et al., '74). After penetration, the original araAMP moiety was phosphorylated to the triphosphate, araATP, and incorporated into DNA.These results provided a biochemical basis for the suggestion that, in certain cases, nucleotides might be more toxic formulations for chemotherapeutic purposes than nucleosides. As nucleotides, the compounds might prevent the degradation of the nucleoside moiety by cellular enzymes or could possibly bypass cellular deficiencies in the enzymes required for activation of the toxic compound. In the former case, we have provided evidence that araAMP and its cyclic derivative, 3', 5'-cyclic araAMP, are more toxic to L-cells than is 9-p-D-arabinofuranosyladenine (araA) (Ortiz et al., '72; Plunkett and Cohen, '75b). In the latter case, we have shown

Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies

“…Even though reverse transcription is more efficient in the presence of detergents, significant quantities of intravirion DNA can be synthesized without detergent and can subsequently augment viral infectivity. It has been suggested that the cellular membrane may not be significantly permeable to either ribonucleoside triphosphates or dNTPs (19,25,29,32). The retroviral envelope is derived from the cellular membrane of virus-producing cells.…”

99mTc-labeled nucleotides as tumor-seeking radiodiagnostic agents.

“…When cells are grown in culture, the increased intracellular levels of these adenine nucleotides result in an arrest of cellular growth and cause a large fraction of the cells to remain in their S phase (1). Treatment of well-characterized human tumor cells in soft agar culture with [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] ,uM ATP yielded substantial inhibition of cellular growth (2).…”

“…Nevertheless, mammalian cells can be infected with low efficiency by viral nucleic acids (5), and mouse fibroblasts (L cells) were shown to be permeable to intact dAMP and araAMP (6), as well as other deoxynucleoside monophosphates (7). Incorporation of low concentrations of nucleotides into cellular pools is impaired by the activity of ectoenzymes such as ATPase, ADPase, alkaline phosphatase, 5'-nucleotidase, and nucleotide pyrophosphatase (8,9).…”