Ronald W. Trewyn

Elevated nucleoside excretion by patients with nasopharyngeal carcinoma: Preliminary diagnostic/prognostic evaluations

Nucleoside excretion was monitored in the urine of patients with nasopharyngeal carcinoma, a difficult malignant disease to diagnose at early stages. Separation and quantitation of nucleosides were accomplished by using high performance liquid chromatography. Significantly elevated nucleoside levels were found for patients with newly diagnosed and recurrent carcinomas. The magnitude and timing of the increases indicate the value of monitoring these levels for diagnostic/prognostic evaluations for nasopharyngeal carcinoma.

Urinary Nucleosides in Leukemia: Laboratory and Clinical Applications

Urinary nucleosides offer a number of useful laboratory and clinical applications in the study and analysis of leukemia. There are significant differences in the excretion of modified nucleosides between normal individuals and individuals with various forms of leukemia, as well as between leukemia patients at opposite ends of the clinical spectrum, i.e., those with active disease and those in remission. The nucleoside excretion levels correlate to bone marrow tumor burden in certain forms of leukemia, and limited serial data indicate the potential value of the nucleosides for predicting relapse before the disease deterioration can be recognized clinically. In addition, it may be feasible to assess the effectiveness of chemotherapy used in the treatment of leukemia much more rapidly with the urinary nucleoside markers than with conventional invasive methods.

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.

Queuine hypomodification of tRNA induced by 7-methylguanine.

Abstract 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 in vitro .

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.

Alterations in tRNA Metabolism as Markers of Neoplastic Transformation

Numerous changes in tRNA modification and catabolism are observed when cells undergo neoplastic transformation. The nature of certain of these changes is well established, while others require further characterization. Enzymes involved in the macromolecular modification of tRNA, the tRNA methyltransferases, exhibit idiosyncratic alterations during neoplastic transformation. These alterations include increases in enzyme specific activity as well as the appearance of different tRNA methyltransferases in the malignant tissue1,2. The increased tRNA methyltransferase activity and capacity observed in vitro for the enzymes isolated from transformed cells can also be correlated to increased methylation of specific tRNA iso accepting species, although not total tRNA, in vivo 3.

6-Ethylmercaptopurine-mediated growth inhibition of HL-60 cells in vitro irrespective of purine salvage

SummaryA variety of purine analogs inhibit the growth and induce the differentiation of human promyelocytic leukemia (HL-60) cells that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Mechanisms by which purine analogs induce differentiation offer unique potential for cancer chemotherapy. The guanine analogs, 6-thioguanine and 8-azaguanine, induce granulocytic differentiation of HGPRT-deficient HL-60 promyelocytes. Although these compounds are useful as model purine analogs that induce differentiation in HGPRT-deficient HL-60 cells, they suffer the disadvantage that they are highly cytotoxic to wild-type cells. We studied the effect of the hypoxanthine analog 6-ethylmercaptopurine on wild-type and HGPRT-deficient HL-60 cells. 6-Ethylmercaptopurine inhibits growth and produces a specific terminal end-cell in both types of HL-60 cells. The mechanism appears to be independent of the normal modes of cytotoxic activation through HGPRT or adenine phosphoribosyltransferase (APRT), since no new peaks were seen in HPLC chromatograms of the nucleotide pools. Furthermore, hypoxanthine and adenine failed to prevent growth inhibition by 6-ethylmercaptopurine, and inhibition of IMP dehydrogenase and the consequential alteration of the guanine nucleotide pools does not appear to be involved. The mechanism differs from that of guanine analog-induced differentiation in HGPRT-deficient HL-60 cells.

Altered growth properties of normal human cells induced by phorbol 12,13-didecanoate

SummaryThe tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cell with 10−7 or 10−8M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4α-phorbol 12,13-didecanoate failed to elicit similar cellular responses.

Hematopoiesis and the Inosine Modification in Transfer RNA

Abstract Human promyelocytic leukemia (HL-60) cells were used to begin to evaluate the role in hematopoiesis of inosine biosynthesis in the tRNA anticodon wobble position; a reaction involving the enzymatic insertion of preformed hypoxanthine. Dimethyl sulfoxide (DMSO) and hypoxanthine were found to induce the differentiation of HL-60 cells in a synergistic manner, and the induced differentiation was independent of changes in the purine catabolic enzymes adenosine deaminase and purine nucleoside phosphorylase. The short-term exposure of HL-60 cells to DMSO plus hypoxanthine resulted in enhanced leucine incorporation, and a model is presented showing how the inosine modification reaction in tRNA may be involved. A means by which hypoxanthine insertion into tRNA may modulate the synthesis of regulatory proteins (e.g., lymphokines and cell surface receptors) is also outlined.

Chapter 5 Site Directed Replacement of Nucleotides in the Anti-Codon Loop of Trna: Application to the Study of Inosine Biosynthesis in Yeast TRNAALA

Publisher Summary This chapter describes recombinant RNA technology which allows generating the required transfer RNA (tRNA) substrates in vitro by replacing specific nucleotides within the anticodon loop. Yeast tRNA Ala with the anticodon IGC was reconstructed to contain the anticodon AGC, and this tRNA was used as a substrate for the A 34 to I 34 modification catalyzed in vitro by the tRNA-hypoxanthine ribosyltransferase from cultured human promyelocytic leukemia cells. The key to utilizing this technology is based on placing a [ 32 P] label adjacent to the nucleoside of interest, so the modification reaction can be monitored. Recombinant RNAs are investigated by employing reconstructed tRNAs to examine specific modification reactions in vitro and in vivo. The site directed replacement of one or more nucleotides in pure tRNA isoacceptors has led to new insights into the mechanism and specificity of several modification enzymes acting at positions 34 and 37 in the anticodon loop, and similar technology should prove useful for future analyses of the nature and role of these important modifications.