Drastic rise of intracellular adenosine(5′)tetraphospho(5′)adenosine correlates with onset of DNA synthesis in eukaryotic cells

An assay of adenosine(5′)tetraphospho(5′)adenosine (Ap₄A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap₄A in cellular extracts. In eukaryotic cells this method yielded levels of Ap₄A varying from 0.01 μM to 13 μM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G₁‐arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap₄A pools gradually increased 1000‐fold during progression through the G₁ phase reaching maximum Ap₄A concentrations of about 10 μM in the S phase. Quiescent 3T3 cells reach a high level of Ap₄A (1 μM) in a “committed” but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap₄A pool. Cells lacking a defined G₁ phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap₄A (about 0.3 μM) even during mitosis. From this high level, Ap₄A concentration increases only about tenfold during the S phase. Temperature‐down‐shift experiments, using chick embryo cells infected with transformation‐defective temperature‐sensitive viral mutants(td‐ts), have shown that the expression of the transformed state at 35°C is accompanied by a tenfold increase of the cellular Ap₄A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap₄A levels within 20 h. The possibility of Ap₄A being a “second messenger” of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap₄A is a ligand of mammalian DNA polymerase α, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.