Diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) levels under various proliferative and cytotoxic conditions in several mammalian cell types

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) has been proposed as an intracellular signal for growth. In order to test this hypothesis Ap4A levels were followed in several cell types under various conditions. Quiescent dog thyroid cells in a primary culture were induced to proliferate by addition of a mixture of epidermal growth factor, thyrotropin and foetal calf serum; V79 cells were synchronized by serum depletion then stimulated to proliferate by addition of foetal calf serum. Protein and DNA synthesis increased in both cases, although no significant changes in Ap4A levels per cell could be demonstrated. HeLa D98/AH2 and L929 cells were treated with human recombinant tumour necrosis factor alpha which caused marked cell death. This was measured by a decrease in DNA content and a release into extracellular medium of incorporated radioactive precursor. No concomitant variations in Ap4A concentrations could be observed under these conditions. The data from these various systems do not support the hypothesis that changes in Ap4A levels regulate cellular proliferation.     

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 microM to 13 microM 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 microM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 microM) 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 microM) 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 degrees 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 alpha, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.     

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Interleukin-2 regulation of diadenosine 5',5'-p1 p4-tetraphosphate (Ap4A) levels and DNA synthesis in cloned murine T lymphocytes

The levels or diadenosine 5', 5'-p1, p4, tetraphosphate (Ap4A), a putative signal molecule associated with DNA synthesis, has been measured in murine T lymphocytes. The level or Ap4A detected correlated with the stimulation of DNA synthesis in murine T lymphocytes. In interleukin-2 (IL-2) dependent cells previously deprived of IL-2, new DNA synthesis can be induced by adding IL-2; the synthesis of DNA is preceded by an increase in Ap4A levels. A significant increase in DNA synthesis was observed after the Ap4A concentration exceeded the Kd of DNA polymerase alpha for Ap4A. Similarly, in cells blocked from synthesizing DNA by hydroxyurea, the levels or Ap4A are maintained only in the presence of IL-2. Once IL-2 is removed, the potential to synthesize DNA decreases and is preceded by decreases in the level or Ap4A. The DNA synthesis potential decreases rapidly after the Ap4A concentration fell below the Kd of DNA polymerase alpha for Ap4A. It is possible that Ap4A is a second messenger molecule required for the proliferation of lymphocytes and that the production of Ap4A in IL-2 dependent murine T lymphocytes is regulated by the homologous growth factor.     

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

Zajdela hepatoma culture cells (ZHC) and mouse embryo fibroblasts (Swiss 3T3) were synchronized in G1 or S phase by serum deprivation and aphidicolin treatment, respectively, to study the variations in adenylyl nucleotide (Ap4X) pool size during the progress of the cell cycle. Only minor variations, which never exceeded a factor of 2, were observed when the Ap4X concentrations were expressed on a cellular basis. The variations were found to be strictly parallel to the ATP variations. Upon release from an aphidicolin block, the minor variations of Ap4X followed DNA synthesis and preceded cytokinesis. When the nucleotide content was compared with the amount of proteins, the faint specific cell cycle changes were almost completely damped when the cells were synchronized by serum deprivation, but remained practically unchanged in the case of aphidicolin synchronization. These results suggest that the observed variations could reflect the accumulation of some nucleotides before cell division. It is not clear yet whether the variation in Ap4X concentration is significant by itself or is simply a phenomenon resulting from changes in the ATP pool.     

Diadenosine 5'',5''"-P1,P4-tetraphosphate in developing embryos of Artemia

Diadenosine 5',5'"-P1,P4-tetraphosphate (Ap4A) has been detected in cysts and developing embryos of the brine shrimp Artemia in amounts 10(4)-10(6) times lower than that of the guanine analogue, Gp4G. The unexpectedly high level of Ap4A in dormant cysts of 2.37 pmol/10(6) cells can be reduced to 0.03 pmol/10(6) cells by decapsulation and storage in saturated NaCl. When development is reinitiated, the Ap4A content of the decapsulated embryos undergoes a rapid 125 -fold increase, reaching a maximum of 3.79 pmol/10(6) cells at the point of emergence when DNA replication begins. If replication is delayed by hypoxia, the Ap4A level is adjusted in order to reach the same maximum value when replication finally begins. As replication proceeds, the level of Ap4A declines again. Unlike mammalian cells, Ap4A in Artemia is less metabolically labile than ATP. These results are consistent with the suggested role of Ap4A in the initiation of DNA synthesis.     

Is Ap4A involved in DNA repair processes?

Hepatoma tissue culture (HTC) cells were incubated in the presence of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to study the variations in the bisnucleosides polyphosphates (Ap4X) pool size. A transient but sensitive accumulation of these compounds is observed; if 3-aminobenzamide (3AB) which is a potent inhibitor of the ADP-ribosyltransferase (ADPRT) is added after the MNNG treatment, a more pronounced and persistent accumulation of Ap4X can be seen. A moderate heat-shock (30 min at 43 degrees C) results also in a small accumulation of Ap4X but the shape of the accumulation curve is quite different and the increase of the Ap4X pool is not sensitive to the presence of 3AB. However, both MNNG treatment and hyperthermia cause a marked inhibition of protein synthesis. On the other hand, the ADPRT activity is enhanced in the presence of MNNG whereas hyperthermia has little or a slightly inhibitory effect on this activity. These results suggest that MNNG treatment triggers an Ap4X accumulation in eukaryotic cells different from that observed after heat-shock and it seems likely that these compounds are involved in the DNA excision repair system in which the ADPRT enzyme is also implicated.     

Accumulation of dinucleoside polyphosphates in Saccharomyces cerevisiae under stress conditions. High levels are associated with cell death

Adenosine tetraphosphonucleosides (Ap4X) were measured in Saccharomyces cerevisiae by a coupled phosphodiesterase-luciferase assay. After exposure of the cells to cadmium or to hyperthermic treatment (46 degrees C) a marked increase of the cellular pool from 0.08 microM (base level) to 4 microM or higher was observed. The accumulation of Ap4X to high levels is associated with irreversible processes leading to cell death.     

P1,P4-Di(adenosine-5')tetraphosphate inhibits phosphorylation of immunoglobulin G by Rous sarcoma virus pp60src

Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 2-6) inhibited phosphorylation of immunoglobulin G from tumor-bearing rabbits (TBR IgG) by pp60src protein kinase purified from Rous sarcoma virus-transformed rat tumor cells. Ap4A, a nucleotide associated with eukaryotic cell proliferation, was one of the most effective inhibitors in the series, causing 50% inhibition of TBR IgG phosphorylation at 15 microM. Ap4A inhibited pp60src-dependent phosphorylation of TBR IgG in solution and immunoprecipitates, as well as the phosphorylation of tubulin, microtubule-associated proteins, and vinculin. Under similar assay conditions, Ap4A did not inhibit phosphorylation of histone H2b by cAMP- or cGMP-dependent protein kinases. Ap4A appears to interact noncovalently with the enzyme, because removal of pp60src by immunoprecipitation from solutions containing Ap4A restored activity to uninhibited levels. A 100-fold increase in ATP (4-400 nM) caused a 13-fold increase in the 50% inhibitory concentration of Ap4A (2.5-33 microM), consistent with the interpretation that Ap4A competes for an ATP-binding site on the pp60src molecule. The simplest explanation of these results is that Ap4A binds to the phosphodonor site for ATP.     

Investigation into the interactions between diadenosine 5',5'-P1,P4-tetraphosphate and two proteins: molecular chaperone GroEL and cAMP receptor protein

Diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A) is a dinucleoside polyphosphate found ubiquitously in eukaryotic and prokaryotic cells. Despite Ap(4)A being universal, its functions have proved to be difficult to define, although they appear to have a strong presence during cellular stress. Here we report on our investigations into the nature and properties of putative Ap(4)A interactions with Escherichia coli molecular chaperone GroEL and cAMP receptor protein (CRP). We confirm previous literature observations that GroEL is an Ap(4)A binding protein and go on to prove that binding of Ap(4)A to GroEL involves a set of binding sites (one per monomer) distinct from the well-known GroEL ATP/ADP sites. Binding of Ap(4)A to GroEL appears to enhance ATPase rates at higher temperatures, encourages the release of bound ADP, and may promote substrate protein release through differential destabilization of the substrate protein-GroEL complex. We suggest that such effects should result in enhanced GroEL/GroES chaperoning activities that could be a primary reason for the improved yields of the refolded substrate protein observed during GroEL/GroES-assisted folding and refolding at >or=30 degrees C in the presence of Ap(4)A. In contrast, we were unable to obtain any data to support a direct role for Ap(4)A interactions with CRP.     

NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line

Overexpression of neuropeptide Y (NPY) and its receptors has been found in various cancers. In our previous study, we demonstrated expression of NPY Y5 receptor (Y5R) in various breast cancer cell lines along with Y1 receptor. In Y5R expressing BT-549 cells, NPY induced cell proliferation that was blocked by Y5R-selective antagonist CGP1683A (CGP). Here, NMR-based metabonomics was used to monitor the metabolic profile of BT-549 cells in the presence of NPY and CGP to assess the effect of Y5R activation and inhibition during NPY-induced cell proliferation. To study changes in intra and extra cellular metabolites in response to various treatments, 1D ¹H-NMR spectra of both hydrophilic cell extracts and growth medium were recorded from BT-549 with three treatments: (1) NPY, (2) CGP, and (3) CGP followed by NPY (CGP/NPY). Principal component analysis and statistical significance analysis indicated changes in intracellular concentrations of seven metabolites in hydrophilic cell extracts with NPY treatment: decreases in lactate, succinate, myo-inositol, and creatine, and increases in acetate, glutamate, and aspartate. A significant increase in intracellular lactate level and attenuation of other metabolites to baseline was detected in CGP/NPY group. Also, significant decreases in lactate and increases in pyruvate were observed in growth medium from NPY treated cells. Based on the metabonomics analysis, Y5R activation induces cell proliferation by increasing the rate of glycolysis, glutaminolysis, and TCA cycle. Inhibition of Y5R by CGP counteracts NPY-induced changes in cellular metabolites. These changes may play a role in cell proliferation and migration by NPY through Y5R activation.     

High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.     

Yeast diadenosine 5',5'-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase

The diadenosine 5',5'-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.     

The binding activities of proteins that bind Ap4A, an alarmone, are stimulated in the presence of ethanol or phosphatidylethanolamine

Three proteins binding Ap4A which is known to increase in the heat-shocked cells or to trigger DNA synthesis in G1-arrested eukaryotic cells were purified from E. coli cell extract. For the binding activities of the proteins, glutathione or dithiothreitol and manganese or iron ion were absolutely required. Glutathione, which exists in relatively high concentration in the cells and had been reported to be related to oxidant shock, was far more effective than an artificial antioxidant, dithiothreitol. Ethanol, which has an effect similar to heat or oxidant shock on microbial or eukaryotic cells, enhanced several fold the Ap4A-binding activity. Phosphatidylethanolamine, a major component of phospholipids of cytoplasma and membrane of E. coli cell also stimulated the Ap4A-binding activity.     

Diadenosine tetraphosphate (Ap4A) is compartmentalized in nuclei of mammalian cells

The intracellular compartmentation of Ap4A in various growth and cell-cycle stages in mammalian cells was studied by applying a non-aqueous extraction procedure for cell nuclei. In both slowly and in exponentially growing Ehrlich ascites tumour cells from random cultures, more than 75% of the whole cellular Ap4A content is localized in the nuclei. In G1 and early S-phase cells of synchronized baby hamster kidney (BHK) fibroblast cultures, approx. 90% of the intracellular Ap4A pool is confined to the nuclear compartment. In contrast, Ap4A is distributed to nearly equal amounts between cytoplasm and nuclei during mid-S phase. After transition through the S-phase, increasing proportions of Ap4A (78% 18 h and 96% 22 h after serum replenishing, respectively) are again localized in the nuclear compartment.     

Characterization of P1,P4-diadenosine 5'-tetraphosphate binding on bovine aortic endothelial cells

In recent years it has become increasingly clear that alpha, omega-dinucleotides act as extracellular modulators of various biological processes. P1,P4-diadenosine 5'-tetraphosphate (Ap4A) is the best characterized alpha,omega-dinucleotides and acts as an extracellular signal molecule by inducing the release of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) (R. H. Hilderman, and E. F. Christensen (1998) FEBS Lett. 407, 320-324). However, the characteristics of Ap4A binding to endothelial cells have not been determined. In this report we demonstrate that Ap4A binds to a heterogeneous population of receptors on BAEC. Competition ligand-binding studies using various adenosine dinucleotides, guanosine dinucleotides, adenosine/guanosine dinucleotides, and synthetic P2 purinoceptor agonists and antagonists demonstrate that Ap4A binds to a receptor on BAEC that has a high affinity for some of the adenosine dinucleotides. The apparent IC50 values for Ap4A, Ap2A, and Ap3A are between 12 and 15 microM, while the apparent IC50 values for Ap5A and Ap6A are greater than 500 microM. Evidence is also presented which suggests that this receptor can be classified as a putative P4 purinoceptor. Competition studies also demonstrate that Ap4A binds at a lower affinity to a second class of binding sites.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Alterations in levels of 5'-adenyl dinucleotides following DNA damage in normal human fibroblasts and fibroblasts derived from patients with xeroderma pigmentosum

Levels of 5'-adenyl dinucleotides, measured as diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A), were found to accumulate in cultured human fibroblasts following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the radiomimetic drug bleomycin, and nitroquinoline-1-oxide (NQO) or UV-irradiation in the presence of cytosine arabinofuranoside (araC). In contrast, cells derived from patients with xeroderma pigmentosum complementation group A (XP-A) did not demonstrate an increase in DNA-strand breaks following UV irradiation or NQO in the presence of araC nor an increase in Ap4A levels. Ap4A accumulation did occur in XP-A cells following treatment with MNNG. Cells derived from patients characterized as XP variants, which are incision repair-proficient, accumulated 5'-dinucleotides following bleomycin, MNNG and UV or NQO in the presence of araC. Taken together, these data suggest that Ap4A accumulates as a response to DNA-strand breaks.     

Significance of dinucleoside tetraphosphate production by cultured tumor cells exposed to the presence of ethanol

The use of 30 to 50% ethanol solutions to extract the nucleotides from HTC and A-459 cells results in dinucleoside tetraphosphate (Ap4X) levels 3-30-fold as high as those obtained by 5% classical trichloracetic acid extraction, while ATP levels are identical in both cases. The amplification factor varies with the percentage of ethanol and duration of contact between the cells and the extraction mixture. It remains constant for the HTC cells during cell growth, but exhibits a maximum for the A-459 cells towards the end of the exponential growth period. The incorporation of radioactivity in Ap4X when [alpha-32P]ATP is added to the extraction mixture suggests an Ap4X neosynthesis in the presence of ethanol. The results carried out in the presence of pyrophosphate, EDTA and zinc acetate strongly suggest that aminoacyl-tRNA synthetases could be responsible for the increase in Ap4A content with ethanol treatment. Nevertheless, the effect of ethanol is probably not the result of an activation of these enzymes, but rather, as already suggested by earlier results in our laboratory, the result of a fast inactivation of the degradation enzymes.     

Di(1,N6-ethenoadenosine)5', 5'-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N6-ethenoadenosine)5',5'-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.