The growth of Sarcoma 180 cells (S-180), whether dependent on the synthesis of purine nucleotides de novo (folate medium) or on preformed purines (amethopterin medium) such as hypoxanthine, adenine, or adenosine, was inhibityed by 50 and 100% at 15-22 and 100 µM N6-(Δ2-isopentenyl)adenosine, respectively. This adenosine analogue was shown to accumulate in the cell water in the form of the 5'-monophosphate at 2 mM when the extracellular nucleoside level was 100 µM. In S-180 cells incubated with 100 and 300 µM N6-(Δ2-isopentenyl)adenosine and 30 µM [8-14C]adenine the formation of [8-14C]ATP was inhibited by 55 and 64%, respectively, and [8-14C]AMP accumulated at 53 µM as compared with the control, where AMP was undetectable. Under similar conditions the conversion of 100 µM [8-14C]hypoxanthine to ATP and GTP was inhibited by 51 and 65%, respectively. A study was made on the effect of chemically prepared N6-(Δ2-isopentenyl)adenosine 5'-monophosphate on several enzymes involved in the metabolism of purine nucleotides, using cellfree extracts of S-180 cells. This compound inhibited adenosine kinase by competing with ATP (Ki/Km, 1.4), adenine phosphoribosyltransferase by competing with 5-phosphoribosyl 1-pyrophosphate (Ki/Km, 4.0), AMP kinase by competing with AMP (Ki/Km, 8.8), IMP dehydrogenase by competing with IMP (Ki/Km, 15), and adenylosuccinate synthetase noncompetitively with respect to IMP (Ki, 3.0 mM). This nucleotide analogue was also a moderate inhibitor of 5-phosphoribosyl 1-pyrophosphate synthetase, and a poor inhibitor of adenylosuccinate lyase, hypoxanthine phosphoribosyltransferase, and guanylate kinase. Since the multiplication of S-180 cells in vitro is independent of preformed purines, it appears that the inhibition of adenine phosphoribosyltransferase or of adenosine kinase cannot be the basis for the cytotoxicity of N6-(Δ2-isopentenyl)adenosine. The intracellular pools of AMP and IMP were normally quite low (much less than 0.1 mM) as compared with that of N6-(Δ2-isopentenyl)adenosine 5'-monophosphate. It appears, therefore, that AMP kinase and IMP dehydrogenase may be the critical sites of action. Inhibition of these enzymatic steps could cause a shortage of ATP and GTP, leading to inhibition of all the synthetic processes requiring these triphosphates and, eventually, to cell death.
- Copyright ©, 1974, by Academic Press, Inc.