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Biomedical subjects

M S Elliott

Publications and source records attributed to M S Elliott.

15 recordsLinked to original sources
PubMed1999

Determination of queuosine modification system deficiencies in cultured human cells.

Queuosine-deficient tRNAs are often observed in neoplastic cells. In order to determine possible sites for malfunction of the multistep queuosine modification system, comprehensive studies were performed on two human neoplastic cell lines, the HxGC(3) colon adenocarcinoma and the MCF-7 breast adenocarcinoma, which are 100 and 50-60% queuosine deficient, respectively. These results were compared with data obtained from normal human fibroblast (HFF) cultures which maintain 100% queuosine-modified tRNA populations. Queuine uptake in all three cell types was similar and each demonstrated activation by protein kinase C (PKC). However, incorporation of queuine into tRNA by tRNA:guanine ribosyltransferase (TGRase; E.C. 2.4.2.24) and PKC-catalyzed activation of this enzyme occurred only in HFF and MCF-7 cells. The HxGC(3) cell line exhibited no TGRase activity as was expected. Treatment with 5-azacytidine (5-azaC) induced TGRase activity to a level 20% of that in HFF and MCF-7 cells; however, this 5-azaC-induced TGRase activity was not regulated by PKC. Salvage of the queuine base from tRNA degradation products has been shown in mammalian cells and was measured in the HFF cells. However, salvage activity in the MCF-7 cell line was deficient. Therefore, it was shown by direct measurements that the HxGC(3) cell line is completely lacking in queuosine-modified tRNA due to loss of functional TGRase, while the MCF-7 cell line has an inefficient queuine salvage mechanism resulting in a significant deficiency of queuosine-modified tRNA. These techniques can be applied to any cultured cell types to determine specific lesions of the queuosine modification system, which have been suggested to be associated with neoplastic progression.

Azacitidine↗
PubMed1999

The effect of queuosine on tRNA structure and function.

Computational modeling was performed to determine the potential function of the queuosine modification of tRNA found in wobble position 34 of tRNAasp, tRNAasn, tRNAhis, and tRNAtyr. Using the crystal structure of tRNAasp and a tRNA-tRNA-mRNA complex model, we show that the queuosine modification serves as a structurally restrictive base for tRNA anticodon loop flexibility. An extended intraresidue and intramolecular hydrogen bonding network is established by queuosine. The quaternary amine of the 7-aminomethyl side chain hydrogen bonds with the base's carbonyl oxygen. This positions the dihydroxycyclopentenediol ring of queuosine in proper orientation for hydrogen bonding with the backbone of the neighboring uridine 33 residue. The interresidue association stabilizes the formation of a cross-loop hydrogen bond between the uridine 33 base and the phosphoribosyl backbone of the cytosine at position 36. Additional interactions between RNAs in the translation complex were studied with regard to potential codon context and codon bias effects. Neither steric nor electrostatic interaction occurs between aminoacyl- and peptidyl-site tRNA anticodon loops that are modified with queuosine. However, there is a difference in the strength of anticodon/codon associations (codon bias) based on the presence or lack of queuosine in the wobble position of the tRNA. Unmodified (guanosine-containing) tRNAasp forms a very stable association with cytosine (GAC), but is much less stable in complex with a uridine-containing codon (GAU). Queuosine-modified tRNAasp exhibits no bias for either of cognate codons GAC or GAU and demonstrates a lower binding energy similar to the wobble pairing of guanosine-containing tRNA with a GAU codon. This is proposed to be due to the inflexibility of the queuosine-modified anticodon loop to accommodate proper positioning for optimal Watson-Crick type associations. A preliminary survey of codon usage patterns in oncodevelopmental versus housekeeping gene transcripts suggests a significant difference in bias for the queuosine-associated codons. Therefore, the queuosine modification may have the potential to influence cellular growth and differentiation by codon bias-based regulation of protein synthesis for discrete mRNA transcripts.

Anticodon↗
PubMed1996

Modulation of queuine uptake and incorporation into tRNA by protein kinase C and protein phosphatase.

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.

Biological Transport↗
PubMed1995

Activation of transfer RNA-guanine ribosyltransferase by protein kinase C.

Transfer RNA-guanine ribosyltransferase (TGRase) irreversibly incorporates queuine into the first position in the anticodon of four tRNA isoacceptors. Rat brain protein kinase C (PKC) was shown to stimulate rat liver TGRase activity. TGRase preparations derived from rat liver have been observed to decrease in activity over time in storage at -20 or -70 degrees C. Contamination of the samples by phosphatases was indicated by a p-nitrophenylphosphate conversion test. The addition of micromolar concentrations of the phosphatase inhibitors sodium pyrophosphate and sodium fluoride into TGRase isolation buffers resulted in a greater return of TGRase activity than without these inhibitors. Inactive TGRase preparations were reactivated to their original activity with the addition of PKC. In assays combining both TGRase and PKC enzymes, inhibitors of protein kinase C (sphingosine, staurosporine, H-7 and calphostin C) all blocked the reactivation of TGRase, whereas activators of protein kinase C (calcium, diacylglycerol and phosphatidyl serine) increased the activity of TGRase. None of the PKC modulators affected TGRase activity directly. Alkaline phosphatase, when added to assays, decreased the activity of TGRase and also blocked the reactivation of TGRase with PKC. Denaturing PAGE and autoradiography was performed on TGRase isolates that had been labelled with 32P by PKC. The resulting strong 60 kDa band (containing the major site for phosphorylation) and weak 34.5 kDa band (containing the TGRase activity) are suggested to associate to make up a 104 kDa heterodimer that comprises the TGRase enzyme. This was corroberated by native and denaturing size-exclusion chromatography. These results suggest that PKC-dependent phosphorylation of TGRase is tied to efficient enzymatic function and therefore control of the queuine modification of tRNA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.2.29). We measured the specific release of queuine from Q-5'-phosphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC3 colon adenocarcinoma, prompted us to examine a HxGC3-derived cell line, GC3/M. GC3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.

Adenocarcinoma↗
PubMed1990

Interferon induced inhibition of queuine uptake in cultured human fibroblasts.

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.

Biological Transport↗
PubMed1990

Protein kinase C modulation of queuine uptake in cultured human fibroblasts.

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Identifying inhibitors of queuine modification of tRNA in cultured cells.

Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells. Monolayer cultures (2cm2) of Chinese hamster embryo cells depleted of queuine for 24 h were evaluated for their ability to incorporate [3H]dihydroqueuine into acid precipitable material (tRNA) in the presence and absence of potential inhibitors. Known inhibitors of the queuine modification enzyme tRNA-guanine ribosyltransferase (e.g., 7-methylguanine, 6-thio-guanine, and 8-azaguanine) were very effective in blocking incorporation of the radiolabel, and the dose-dependent results exhibited small standard deviations in independent experiments. The data indicate that the method is rapid, reliable, and potentially useful with a variety of cell types.

Animals↗
PubMed1986

Inhibition of queuine uptake in diploid human fibroblasts by phorbol-12,13-didecanoate. Requirement for a factor derived from early passage cells.

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.

Cells, Cultured↗
PubMed1985

Inhibition of queuine uptake in cultured human fibroblasts by phorbol-12,13-didecanoate.

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.

Biological Transport↗
PubMed1984

Inosine biosynthesis in transfer RNA by an enzymatic insertion of hypoxanthine.

An enzyme was discovered which incorporates hypoxanthine into mature tRNA macromolecules. This enzyme is postulated to be similar to tRNA-guanine ribosyltransferase which inserts 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl )-7-deazaguanine into the first position of the anticodon of four tRNAs. The hypoxanthine-incorporating enzyme has been assayed in extracts of rat liver and cultured human leukemia cells and it has been resolved from tRNA-guanine ribosyltransferase by DEAE-cellulose column chromatography. The enzyme assay is based on the incorporation of radiolabeled hypoxanthine into unfractionated heterologous tRNA and the reaction rate is proportional to the amount of added enzyme extract. Hydrolysis of the radiolabeled tRNA and analysis of the nucleoside composition yields inosine (the nucleoside of hypoxanthine) as the only radiolabeled product. It is proposed that the enzyme, a tRNA-hypoxanthine ribosyltransferase, is responsible for the biosynthesis of inosine in the anticodon wobble position of specific tRNAs, resulting in greatly expanded codon recognition by these tRNAs.

Animals↗
PubMed1984

Relationship between a tumor promoter-induced decrease in queuine modification of transfer RNA in normal human cells and the expression of an altered cell phenotype.

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.

Carcinogens↗
PubMed1981

Interaction of 2-aminobicyclo[3.2.1]octane-2-carboxylic acid with the amino acid transport systems of the sarcoma 37 murine ascites tumor cell.

The relatively broad and overlapping specificities of amino acid transport systems have made the synthesis of analogues specific to single transport systems desirable. The analogue in general use as a specific substrate for transport system L has been 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH). The affinity of BCH for the binding site of system L has been shown to be less than that of the natural substrate, leucine. Earlier studies from this laboratory suggested that higher homologues in a series could have greater affinity for system L. A higher homologue of BCH, 2-aminobicyclo[3.2.1]octane-2-carboxylic acid (ABOCA), has been synthesized and studied as a substrate and competitor for amino acid transport systems of the sarcoma 37 (S37) ascites cell. ABOCA inhibited the transport system dominant in the low concentration region for histidine uptake (system L) but had no effect on the uptake of labeled N-methyl-alpha-aminoisobutyric acid (MeAIB). MeAIB had no effect on labeled ABOCA uptake in S37 cells. ABOCA inhibited the uptakes of labeled leucine and labeled BCH competitively. Leucine, histidine, and BCH inhibited the uptake of labeled ABOCA competitively. Typical L system substrates demonstrated exchange effects with labeled ABOCA. The b isomer of ABOCA demonstrated slightly greater affinity for system L than did the a isomer. We conclude that ABOCA is an analogue restricted to interaction with amino acid transport system L, that it has greater affinity for system L than does BCH, and that its selection for system l is determined principally by an apolar interaction with steric considerations secondary.

Amino Acids↗
PubMedDate not supplied

Queuosine modification of tRNA: a case for convergent evolution.

Queuosine is a hypermodified nucleoside found in position 34, the anticodon wobble position, of four tRNA species. This modification is distributed with near uniformity across all life forms found on this planet. Yet the molecular mechanisms involved with accomplishing this ubiquitous posttranscriptional modification of tRNA are dramatically different between prokaryotic and eukaryotic organisms, which suggests that these were formed by convergent evolution of a fundamental life process essential to nearly all life forms. This minireview describes the differences between these modification systems and points to a new direction for developing research on the molecular function queuosine-modified tRNA in diverse species.

Animals↗