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

Abstract

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.

Cell cycle variations of dinucleoside polyphosphates in synchronized cultures of mammalian cells.

“…A major difference exists between our results and the ones reported by Weinmann-Dorsch et al (30), showing a huge burst (1,000-fold) of Ap4X at the G1-S boundary. It must be pointed out that the extreme Ap4X concentrations reported by Grummt and co-workers (30) are very different from our ones at both the high and low levels.…”

“…A 1,000-fold increase of the intracellular level of Ap4A in baby hamster kidney fibroblasts and mouse 3T3 cells after mitogenic induction of the cells arrested in G1 phase was reported (30). Similarly, in the autosynchronous organism, Physarum polycephalum, a cyclic 10-fold change in Ap4A concentration throughout the cell cycle was described (31).…”

Zinc as a second messenger of mitogenic induction

“…A favorite candidate for such a signal molecule is adenosine(5')tetraphospho(5')adenosine (Ap~). This purine nucleotide has been suggested, on the basis of its high metabolic lability and the correlation of its pool sizes with the growth-and cell cycle state of eukaryotic cells, to be part of the intracellular signal chain of mitogenic induction [5][6][7][8]. This hypothesis was strongly supported by the obser-vation that the site of its intracellular activity is the DNA replicative machinery.…”

“…Since Ap4A levels fluctuate correlated with the growth and cell cycle state of BHK cells [6] we addressed the question whether the effects of EDTA and Zn 2 + on the DNA-synthesizing activity could be mediated by Ap~. Therefore, we studied the effects of Zn 2 + withdrawal by EDTA and its reversal by exogenous Zn 2 + on intracellular Ap4A levels.…”

Regulation of rat mesangial cell growth by diadenosine phosphates.

“…Both compounds are potent coronary and mesenterial vasodilators (8,9). Furthermore, changes in the intracellular concentration of diadenosine tetraphosphate may be involved in cellular proliferation (10)(11)(12). As to the type of receptors, there are divergent findings in literature.…”