phorbol-12-13-didecanoate and queuine

It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that protein phosphatase activity in the cell acts to reverse this. Regardless of the modulation of uptake rate, the level of intracellular queuine base saturates in 6 h. However, there is still an effect on the incorporation rate of queuine into tRNA of fibroblast cultures even after 24 h. We now show that the incorporation of queuine into tRNA in cultured human fibroblasts by tRNA-guanine ribosyltransferase (TGRase) is also stimulated by activators of PKC and inhibitors of protein phosphatase; while inhibitors of PKC decrease the activity of this enzyme. These studies suggest that PKC supports both the cellular transport of queuine and the activity of TGRase in cultured human fibroblasts, and that protein phosphatase activity in fibroblasts acts to reverse this phenomenon. A kinase-phosphatase control system, that is common to controlling both intracellular signal transduction and many enzyme systems, appears to be controlling the availability of the queuine substrate and the mechanism for its incorporation into tRNA. Since hypomodification of transfer RNA with queuine is commonly observed in undifferentiated, rapidly growing and neoplastically transformed cells, phosphorylation of the queuine modification system may be a critical regulatory mechanism for the modification of tRNA and subsequent control of cell growth and differentiation.

Topics: Biological Transport; Carcinogens; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Guanine; Humans; Phorbol Esters; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; RNA, Transfer

Interferon inhibits uptake of the radiolabeled queuine analog, rQT3, into cultured human fibroblasts. Simultaneous exposure to 10 nM phorbol-12,13-didecanoate (PDD) potentiates interferon-induced inhibition of rQT3 into cultured fibroblasts. All three major classes of human interferon tested affected uptake similarly, with fibroblast derived beta-interferon being more effective in dose response than gamma or alpha interferons. This suggests that endogenous production of interferon by cultured cells, such as that observed during a low grade viral infection, inhibits queuine uptake and may subsequently lead to a decreased level of queuine modified transfer RNA. Queuine-hypomodified transfer RNA has been implicated in growth control, differentiation and neoplastic transformation.

Topics: Biological Transport; Dose-Response Relationship, Drug; Fibroblasts; Guanine; Humans; Interferon Type I; Interferon-gamma; Interferons; Phorbol Esters; Poly I-C; Recombinant Proteins

Protein kinase C modulates the activity of a highly specific uptake mechanism for queuine in cultured human fibroblasts. Activators of protein kinase C induce an increased uptake rate for the radiolabeled analog of queuine, rQT3. The protein kinase C inhibitors, H-7, staurosporine and sphingosine all induced a dramatic decrease in the uptake rate of rQT3. This suggests that protein kinase C is tied to efficient cellular uptake of queuine. Uptake is prerequisite to the modification of transfer RNA with queuine. Perturbation of queuine-modified transfer RNA levels has been associated with neoplastic transformation, differentiation and growth control.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Calcimycin; Cells, Cultured; Diglycerides; Enzyme Activation; Fibroblasts; Growth Substances; Guanine; Humans; In Vitro Techniques; Isoquinolines; Male; Phorbol Esters; Phosphatidylserines; Piperazines; Protein Kinase C; Sphingosine; Staurosporine

Cell cultures derived from human neonatal foreskins (HF cells) are susceptible to phorbol-12,13-didecanoate- (PDD) induced inhibition of queuine uptake, but this inhibition is pronounced only in early passage HF cells. The present analysis of five different primary cultures demonstrated that, between 10 and 30 population doublings beyond the primary cultures, HF cells gradually became refractile to PDD-induced inhibition of queuine uptake, after which PDD begins to stimulate queuine uptake. Treating late passage HF cells with conditioned medium from early passage HF cells partially restored the PDD-induced inhibition of queuine uptake. This indicates the existence of a factor produced by early passage HF cells that permits PDD to inhibit queuine uptake. The tumor promoter, teleocidin, mimics the effects of PDD on queuine uptake. Both PDD and teleocidin are known to activate protein kinase C; therefore, this kinase may be an intermediary in tumor promoter-induced effects on queuine uptake. Epidermal growth factor, platelet-derived growth factor, and transforming growth factor beta stimulated queuine uptake in both early and late passage HF cells. Growth factor stimulation of uptake was enhanced by PDD in late passage cells but inhibited by PDD in early passage cells. Polyinosinic polycytidylic acid treatment of late passage HF cells partially restored PDD-induced inhibition of queuine uptake. Human recombinant beta-interferon, plus or minus PDD, had no effect on queuine uptake. PDD did not inhibit queuine uptake in the immortal human and non-human cell lines examined.

Topics: Cells, Cultured; Epidermal Growth Factor; Fibroblasts; Guanine; Humans; Kinetics; Lyngbya Toxins; Peptides; Phorbol Esters; Platelet-Derived Growth Factor; Poly I-C; Transforming Growth Factors

The modified base queuine is inserted posttranscriptionally into the first position of the anticodon of tyrosine tRNA, histidine tRNA, asparginine tRNA, and aspartic acid tRNA. Phorbol-12,13-didecanoate (PDD) effects a decrease in the queuine content of tRNA in cultured human foreskin fibroblasts. The present data suggest that this results from a PDD-mediated inhibition of queuine uptake. Nonsaturable uptake was observed for tritiated dihydroqueuine (rQT3) for up to 2 hr at 10 to 1000 nM concentrations, while saturation of uptake was observed after 3 to 4 hr. Lineweaver-Burke analysis of concentration versus uptake revealed biphasic uptake kinetics with high and low Km components of approximately 350 and 30 nM, respectively. Competition by queuine of rQT3 uptake indicated that both compounds have equal affinity for the uptake mechanism. PDD inhibited rQT3 uptake but required 30 to 60 min of exposure before the uptake was completely blocked. The rQT3 efflux rate from cells was found to be 3 to 4 times greater than that of uptake, and PDD also inhibited the efflux reaction. The potential inhibitors furosemide, nitrobenzylthioinosine, ouabain, 7-methylguanine, 7-deazaguanine, guanine, guanosine, adenine, adenosine, hypoxanthine, and epidermal growth factor had no effect on rQT3 uptake. However, dipyridamole was immediately effective at reducing rQT3 uptake.

Topics: Biological Transport; Carcinogens; Cells, Cultured; Fibroblasts; Guanine; Humans; Kinetics; Phorbol Esters; Phorbols; RNA, Transfer; Tritium

With normal human skin fibroblasts in culture, a transient decrease in queuine modification of tRNA precedes a phorbol ester tumor promoter-induced 5- to 10-fold increase in saturation density. Subsequently, an increase in the queuine content of cellular tRNA (to levels comparable to those in untreated cultures) precedes a decrease in saturation density. This reversal of the phorbol ester-induced alteration in tRNA modification occurs in the continued presence of the tumor promoter, and it parallels an increased ability of the cells to salvage queuine from catabolized endogenous tRNA. Addition of exogenous queuine concurrently with the tumor promoter at early passage significantly inhibits the increase in saturation density. The results suggest a role for the decrease in queuine modification of tRNA in mediating the phenotypic change induced by the tumor promoter.

Topics: Carcinogens; Cells, Cultured; Fibroblasts; Guanine; Humans; Infant, Newborn; Kinetics; Male; Phenotype; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; RNA, Transfer; Skin; Tritium