mRNAs containing the histone 3' stem-loop are degraded primarily by decapping mediated by oligouridylation of the 3' end.

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5'→3' and 3'→5' processes, but the relative contributions are not known. The 3' end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3' UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5'→3' decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5' decay intermediates were oligouridylated. Preventing oligouridylation by 3'-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3'→5' degradation machinery stalls during initial degradation, whereupon reuridylation occurs.

Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling.

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.

Enzyme pattern-directed chemotherapy: synergistic interaction of 3-deazauridine with D-galactosamine.

We tested an experimental approach in which the specialized enzymatic pattern characteristic of the tissue of origin of a tumor might be exploited to target and enhance drug selectivity. In the present work, the D-galactosamine-induced depletion of uridine 5'-triphosphate (primarily a hepatic event) was employed to enhance the growth inhibition caused by 3-deazauridine. As predicted, the drug effect was most pronounced in the slower growing, well differentiated hepatoma lines where the activities of certain hepatic metabolic pathways and enzymes, though decreased, were still operative. The interactions of D-galactosamine and cytosine arabinoside with 3-deazauridine were examined in vitro in four liver tumor cell lines and two nonhepatic lines. The effects of D-galactosamine and 3-deazauridine on the growth of the Morris hepatoma cell lines 3924A, 8999S,AND 8999R were strongly synergistic; on the Novikoff hepatoma and the nonhepatic cell lines they were only additive. The combination of 3-deazauridine with cytosine arabinoside gave approximately additive growth inhibition with all cell types, without selective toxicity towards the hepatocellular lines. Results of growth-inhibition studies with the combination of D-galactosamine and cytosine arabinoside and with combinations of all three agents are also presented. These results are analyzed in the context of the regulation of hepatic pyrimidine nucleotide metabolism and our design of enzyme pattern directed drug selectivity.

Extracellular uridine nucleotides initiate cytokine production by murine dendritic cells.

While it is recognized that activated dendritic cells perform their immune functions with greater efficacy, it is not altogether clear what factors are responsible for such activation. Recent evidence points to an important role for extracellular nucleotides in the modulation of leukocyte function. In the present study we investigated the ability of extracellular nucleotides to activate CD11c(+) murine dendritic cells. Mobilization of intracellular calcium was observed following treatment of these cells with UTP or UDP, but not ATP. Furthermore, this nucleotide receptor was pertussis toxin-sensitive, suggesting the presence of a P2Y nucleotide receptor. Such receptors were not present on murine peritoneal macrophages or on CD11c-negative leukocyte populations. Importantly, activation of these P2Y nucleotide receptors on dendritic cells provided a potent stimulus for cytokine mRNA expression and secretion. Thus, expression of a P2Y nucleotide receptor on CD11c(+) dendritic cells functions to mobilize intracellular calcium and to induce cytokine production.