Guanine analog-induced differentiation of human promyelocytic leukemia cells and changes in queuine modification of tRNA.

Treatment of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient human promyelocytic leukemia (HL-60) cells with 6-thioguanine results in growth inhibition and cell differentiation. 6-Thioguanine is a substrate for the tRNA modification enzyme tRNA-guanine ribosyltransferase, which normally catalyzes the exchange of queuine for guanine in position 1 of the anticodon of tRNAs for asparagine, aspartic acid, histidine, and tyrosine. During the early stages of HGPRT-deficient HL-60 cell differentiation induced by 6-thioguanine, there was a transient decrease in the queuine content of tRNA, and changes in the isoacceptor profiles of tRNA(His) indicate that 6-thioguanine was incorporated into the tRNA in place of queuine. Reversing this structural change in the tRNA anticodon by addition of excess exogenous queuine reversed the 6-thioguanine-induced growth inhibition and differentiation. Similar results were obtained when 8-azaguanine (another inhibitor of queuine modification of tRNA that can be incorporated into the anticodon) replaced 6-thioguanine as the inducing agent. The data suggest a primary role for the change in queuine modification of tRNA in mediating the differentiation of HGPRT-deficient HL-60 cells induced by guanine analogs.     

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.     

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.     

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 critical regulatory mechanism for the modification of tRNA and subsequent control of cell growth and differentiation.     

Protein synthesis in HL-60 cells treated with DMSO and hypoxanthine.

Short-term treatment of the HL-60 cells with DMSO and hypoxanthine, inducers of granulocytic differentiation, was reported to cause a rapid increase in protein synthesis. This effect was ascribed to the insertion of inosine in the wobble position of the tRNA anticodon and consequently increasing codon recognition potential. In this study we have re-investigated the effects of DMSO and/or hypoxanthine on protein synthesis. In contrast to their findings we were unable to demonstrate stimulated protein synthesis in either short- or long-term treatment with these agents. Polysome analysis under these conditions revealed that polysomes were disaggregated. Finally, the activity of tRNA-hypoxanthine ribosyltransferase, an enzyme responsible for the insertion of inosine in the anticodon, was also relatively low. Under these circumstances, we propose that tRNA modification is not essential in the regulation of protein synthesis.     

Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase

We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials. Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position. In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme. Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme. While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor. The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine. The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp. With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.     

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.     

Modeling the Structure of Human tRNA-Guanine Transglycosylase in Complex with 7-Methylguanine and Revealing the Factors that Determine the Enzyme Interaction with Inhibitors

Biochemistry (Moscow) - tRNA-guanine transglycosylase, an enzyme catalyzing replacement of guanine with queuine in human tRNA and participating in the translation mechanism, is involved in the...     

Synthesis of tetrahydrobiopterin in friend erythroleukemia cells and its modulator effect on cell proliferation

The induction of the enzymes in the tetrahydrobiopterin pathway by dimethyl sulfoxide (DMSO) was investigated in subclones F4N and B8/3 of the proerythroblastoid Friend erythroleukemia cell line (MEL). GTP-cyclohydrolase, the initial enzyme in the biosynthetic pathway, is virtually absent in both clones, but expression increases during 3 days of DMSO treatment. The final enzyme levels show 12-fold (subclone B8/3) and 40-fold (subclone F4N) increases compared to initial values. Enhancement of 6-pyruvoyl tetrahydropterin synthase activity is detectable 6 h after exposure to DMSO and continues to increase in the 3-day time period to 2.4-fold and 1.8-fold levels in subclones B8/3 and F4N, respectively. Sepiapterin reductase is present in unstimulated F4N cells and absent in B8/3 cells. The enzyme activity is not affected by DMSO treatment in either cell line. This explains why DMSO treatment causes accumulation of tetrahydrobiopterin in the MEL subclone F4N, but not in subclone B8/3. MEL cells are devoid of phenylalanine hydroxylase for which tetrahydrobiopterin serves as cofactor. In F4N, but not in B8/3, tetrahydrobiopterin modulates the rate of [3H]thymidine incorporation, thus being functionally linked with cell proliferation rather than with differentiation. In contrast to T lymphocytes, periods of tetrahydrobiopterin synthesis and of modulator function are uncoupled in MEL cells.     

Queuine metabolism and cadmium toxicity in Drosophila melanogaster

Queuine can replace guanine in the anticodon of certain tRNAs and is a hypermodified guanine derivative that can be synthesized by bacteria but not by mice. The study demonstrates that Drosophila can incorporate dietary queuine into tRNA but cannot synthesize it de novo for this purpose. Since an earlier study had shown that dietary CdCl2 caused Drosophila to increase greatly the proportion of queuine-containing tRNA over non-queuine tRNA the ability of dietary queuine to counteract cadmium toxicity was evaluated. When queuine was present in the cadmium-containing medium more pupae matured into adults than when queuine was absent. Other studies had demonstrated that the transglycosylase enzyme, that catalyzes the replacement of guanine in the anticodon of tRNA by queuine, is present in Drosophila larvae but the tRNA is virtually devoid of queuine. This study shows that in the presence of dietary queuine the larval tRNA contains abundant amounts of queuine. Therefore, we postulate a significant role for bacteria in supplying queuine to Drosophila for its incorporation into tRNA and that the control of this process by Drosophila is passive, i.e. is not an essential feature in differentiation.     

Modulation of Lactate Dehydrogenase Isozymes by Modified Base Queuine

The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA_GUN) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.     

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.     

Queuine hypomodification of tRNA induced by 7-methylguanine

Transfer RNA isolated from Chinese hamster cells transformed by 7-methylguanine is hypomodified for queuine. 7-Methylguanine rapidly induces queuine hypomodification of tRNA in normal Chinese hamster embryo cells under conditions leading to transformation, and the enzyme catalyzing the queuine modification reaction, tRNA: guanine transglycosylase, is inhibited by 7-methylguanine $\text{in}\text{vitro}$.     

tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.     

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase to elucidate the role of serine 103 for enzymatic activity.

The tRNA modifying enzyme tRNA-guanine transglycosylase (TGT) is involved in the exchange of guanine in the first position of the anticodon with preQ1 as part of the biosynthesis of the hypermodified base queuine (Q). Mutation of Ser90 to an alanine in Escherichia coli TGT leads to a dramatic reduction of enzymatic activity (Reuter, K. et al. (1994) Biochemistry 33, 7041-7046). To further clarify the role of this residue in the catalytic center, we have mutated the corresponding Ser103 of the crystallizable Zymomonas mobilis TGT into alanine. The crystal structure of a TGT(S103A)/preQ1 complex combined with biochemical data presented in this paper suggest that Ser103 is essential for substrate orientation in the TGT reaction.     

Friend erythroleukemia cell differentiation: induction by retinoids

Abstract. Growth in the presence of retinoids was found to induce erythroid differentiation in Friend murine erythroleukemia (MEL) cells in culture. The program of differentiated functions expressed by retinoid-treated cells was quite similar to that promoted by other inducers of MEL cell differentiation. For example, 70% or more of induced cells synthesized hemoglobin which accumulated to a level of 8 μg–10 μg per 10⁶ cells. The level of acetylcholinesterase activity increased two to five-fold in induced cells, and induction by retinoids, like induction by dimethylsulfoxide (DMSO), promoted the appearance of cell surface lumps or 'blebs'. All-trans retinaldehyde, which promoted maximum hemoglobin and acetylcholinesterase synthesis at a concentration of 5 × 10⁻⁷ M, was found to be a more potent inducer than all-trans retinoic acid or retinol, which both showed maximum induction at 1 × 10⁻⁵ M. Like differentiation promoted by DMSO, retinoid-induced differentiation was inhibited by 10⁻⁷ M dexamethasone.