Biomedical subjects

R W Trewyn

Publications and source records attributed to R W Trewyn.

At least 19 recordsLinked to original sources

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↗

PubMed1991

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of the tumor marker 1-methylinosine in human urine.

A highly sensitive enzyme-linked immunoassay (ELISA) was developed to detect and quantify the tumor marker, 1-methylinosine (m1I), in human urine. The rabbit antisera was highly specific for m1I with negligible or no inhibition by other nucleosides excreted into urine. Using the competitive ELISA, nanogram amounts of m1I were easily measured directly in urine. The assay agreed with our previous hplc analysis of m1I in urine for identifying those individuals with chronic myelogenous leukemia. Thus, this assay should greatly facilitate the quantitation of m1I as a tumor marker.

Antibody Specificity↗

PubMed1991

Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.

Anticodon↗

PubMed1990

Modification of the anticodon wobble position of tRNA(Ala) in vitro does not require 5' or 3' processing.

Maturation of eukaryotic tRNA molecules requires nuclear processing as well as nuclear and cytoplasmic modification of specific nucleotides. Nucleotide modifications within the anticodon are found in the majority of all tRNAs and are among the last maturation events to occur in vivo. We show that 5' and 3' processing of SP6 polymerase-generated transcripts are not necessary for the in vitro modification of A----I in the anticodon of tRNA(Ala).

Animals↗

PubMed1990

Noncorrelative c-myc and ras oncogene expression in squamous cell carcinoma cells with tumorigenic potential.

The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface-associated antigens. Increased levels of H-ras mRNA and p21 protein were present in six tumors, but enhanced c-myc mRNA expression was observed in just two tumors. The distribution of oncogene mRNA and SCC antigen-positive cells was not uniform throughout the tumor. Isolation of cells from the tumors was accomplished by cell culture, growth in soft agar, and growth in the nude mouse. One nontumorigenic immortalized cell line, SCC-83-01-82, isolated by passage through soft agar, was treated with 50 micrograms/ml of methyl methane sulfonate (MMS). These MMS-converted cells subsequently expressed a tumorigenic phenotype. In situ hybridization of the tumors that developed in nude mice revealed increased c-myc and H-ras mRNA expression. Serial passage of the MMS-converted tumors in vivo was accompanied by consistent enhanced c-myc expression. However, the levels of H-ras and keratin mRNA expression decreased with passage in vitro. Northern blot analysis of c-myc and H-ras mRNA levels from the original SCC cell line showed no change in expression following MMS treatment. The data suggest that SCC-83-01-82 is a premalignant cell line established from a mixed cell population in the tumor mass. It can be converted to a malignant phenotype by treatment with MMS, and the persistence of malignancy is under molecular control other than changes in the level of c-myc and ras gene expression.

Animals↗

PubMed1990

Differential effect of 6-ethylmercaptopurine on c-myc expression in wild-type and HGPRT-deficient HL-60 cells.

A variety of compounds inhibit the growth and induce differentiation of human promyelocytic leukemia (HL-60) cells. HL-60 subclones that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) can also be induced to differentiate with purine analogs. Mechanisms by which purine analogs induce differentiation offer unique possibilities for cancer chemotherapy. We have studied the effect of the purine analog 6-ethylmercaptopurine (e6MP) on the growth and induction of differentiation in both wild-type and HGPRT-deficient HL-60 cells. We have previously shown that e6MP inhibits cell growth in both wild-type and HGPRT-deficient HL-60 cells without activation through salvage pathways. In this report we evaluate the effect of e6MP on c-myc mRNA expression. c-Myc mRNA, which is amplified in HL-60 cells, has been shown to play a role in the induction of granulocytic differentiation in HL-60 cells. e6MP transiently down-regulates c-myc mRNA in wild-type cells but has no effect on c-myc mRNA expression in HGPRT-deficient HL-60 cells. Despite the differential effects of e6MP on c-myc mRNA, both wild-type and HGPRT-deficient HL-60 cells appear to engage in terminal differentiation. The morphological changes and nonspecific esterase activity induced by e6MP suggest differentiation down the monocytic pathway. However, early monocytic markers such as the rapid induction of c-fos and the stabilization of c-fms mRNA are not observed. In addition, e6MP inhibits TPA-induced monocytic/macrophage differentiation as characterized by stabilization of c-fms mRNA and cellular adherence.

Cell Differentiation↗

PubMed1990

Nontumorigenic squamous cell carcinoma line converted to tumorigenicity with methyl methanesulfonate without activation of HRAS or MYC.

Plasticity of human tumor populations could account for the reason why many tumorigenic human cell lines lose this feature when grown in culture. Methyl methanesulfonate (MMS) was used to convert premalignant squamous cell carcinoma (SCC) cell line SCC-83-01-82 to a malignant phenotype. The MMS-treated SCC-83-01-82 cells (MMS-SCC-83-01-82) produced progressively growing tumors in 5 of 11 splenectomized BALB/c nude mice within 3-5 months. A cell line, designated SCC-83-01-82 CA, was established in vitro from one of the mouse tumors and was repassaged successively. This SCC-83-01-82 CA cell line was aggressively tumorigenic. A tumor greater than or equal to 2.0 cm in size was present within a month, as opposed to the 3-5 months required for the tumors produced by the MMS-SCC-83-01-82 cells. Examination of frozen cross sections by in situ hybridization revealed that focal areas of the tumor produced by the MMS-SCC-83-01-82 cells expressed MYC and HRAS mRNA. However, by the third passage in vivo, the levels of expression of the corresponding genes in the mouse tumors were undetectable. Blot-hybridization analysis of the RNA from the MMS-SCC-83-01-82 cells and the subsequently derived tumors and cells did not indicate any consistent overexpression of MYC, HRAS, or KRAS. Restriction fragment length polymorphism analysis of both MYC and HRAS genes revealed neither rearrangement nor amplification of MYC nor point mutation in the 11th or 12th codon of HRAS. The data suggest that alterations in MYC and HRAS were not directly involved in either the initial transformation or MMS-induced tumorigenic conversion of the SCC-83-01-82 cell line. Persistence of tumorigenicity after reisolation of the MMS-converted premalignant SCC-83-01-82 cells did not disappear immediately following the treatment with MMS.

Animals↗

PubMed1989

Altered queuine modification of transfer RNA involved in the in vitro transformation of Chinese hamster embryo cells.

Altered queuine modification of tRNA has been correlated to neoplastic transformation, but no direct cause and effect relationship has been defined. In the present study, a potential role for this alteration has been assigned. The tRNA in normal Chinese hamster embryo cells is significantly more queuine modified than the tRNA in their transformed Chinese hamster embryo counterparts, even though the specific activity of the queuine modification enzyme is much lower in Chinese hamster embryo cells than in transformed Chinese hamster embryo cells. Substrate availability appears to be responsible for the queuine hypomodification of tRNA in the transformed cells, since addition of excess exogenous queuine to the culture medium results in incorporation of queuine into the anti-codon of the undermodified tRNAs. Most importantly, the excess queuine inhibits anchorage-independent growth of transformed Chinese hamster embryo cells, thereby implicating queuine hypomodification of tRNA in the expression of this transformed phenotype.

Animals↗

PubMed1989

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

A 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.

Antimetabolites, Antineoplastic↗

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↗

PubMed1987

Enhancement of the chemical transformation of Chinese hamster embryo cells in vitro by 7-methylguanine.

The RNA catabolite 7-methylguanine has been shown to inhibit queuine modification of tRNA in Chinese hamster embryo cells under conditions leading to in vitro transformation. Phorbol ester tumor promoters also induce queuine hypomodification of tRNA in normal human cells, and this effect was reported to be correlated directly to the appearance of an altered (transformed) cell phenotype. Based on this common macromolecular alteration, 7-methylguanine was evaluated for its ability to enhance the chemically induced transformation of cultured cells. Two-stage initiation-promotion experiments were undertaken with Chinese hamster embryo cells in vitro to compare the effects of 7-methylguanine to known tumor promoters subsequent to initiation with 3-methylcholanthrene. 7-Methylguanine was able to increase significantly the expression of type III foci as well as anchorage-independent growth, thereby confirming that it can act as a promoting agent in vitro. Methylated guanines that do not induce queuine hypomodification of tRNA were not capable of enhancing these characteristics of in vitro transformation. The results suggest that 7-methylguanine may be a natural, endogenous promoting agent, and that changes in queuine modification of tRNA may play a fundamental role in the promotion of carcinogenesis.

Animals↗

PubMed1987

Inhibition of phorbol ester-mediated phenotypic changes in cultured cells by hypoxanthine.

Hypoxanthine induces the differentiation of certain transformed cells in vitro, so analyses were undertaken to determine whether this purine metabolite might influence the expression of transformed phenotypes induced in normal cells by chemical agents. Chinese hamster embryo cells and human skin fibroblasts in culture were treated with the promoting agent phorbol-12,13-didecanoate (PDD) with or without prior treatment with 3-methylcholanthrene (MCA), and various phenotypic effects were monitored. Hypoxanthine was found to inhibit significantly the formation of type III foci and the increase in saturation density observed for Chinese hamster cells treated with MCA plus the phorbol ester. Inosine and the hypoxanthine analogue allopurinol could also mediate the effect on saturation density, while xanthosine could not. An increase in the saturation density of human skin fibroblasts, which can be induced by the phorbol ester alone, was also inhibited by hypoxanthine. There was no significant effect on the growth rate or the intracellular nucleotide pools with hypoxanthine-treated cells. The results suggest that a normal purine metabolite, hypoxanthine, can modulate the expression of transformed phenotypes induced in vitro by the known tumor promotor PDD. These observations could help in elucidating the cellular basis for promotion of carcinogenesis.

Animals↗

PubMed1987

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.

Antibodies, Monoclonal↗

PubMed1986

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.

Acute Disease↗

PubMed1985

Hematopoiesis and the inosine modification in transfer RNA.

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 performed 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.

Adenosine Deaminase↗

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

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

The 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 cells with 10(-7) or 10(-8) M 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 4 alpha-phorbol 12,13-didecanoate failed to elicit similar cellular responses.

Carcinogens↗