Sylvia J. Kerr

Atypical Transfer RNA's And Their Origin In Neoplastic Cells

Publisher Summary This chapter focuses on the structure and synthesis of transfer RNA (tRNA) and discusses the biochemistry of tRNA methylases. Translation is the most complex molecular mechanism known in the living cell. It requires messenger RNA, which is the simplest of the structures involved, even though it is laden with information. The ribosomes are complex structures composed of RNA and at least a score of different proteins. Transfer RNA is the most complex biomacromolecule known. In addition to these major components, there are ancillary ones— enzymes that transfer amino acids to the transfer RNA. Moreover, there are at least 8 soluble protein factors needed for the completion of the synthesis of a protein. Any or all of these numerous components could be a regulatory factor in protein synthesis. There are three different lines of biological evidence that point to transfer RNA as a regulatory factor. Transfer RNA is a pivotal molecule in protein synthesis. It is the link between the amino acids and the message-bearing nucleic acids. The tRNA methylases are a complex family of enzymes that modify the structure of preformed tRNA by the insertion of methyl groups into specific positions in the four main bases of tRNA. The enzymes are species-specific, organ-specific, base-specific, and even site-specific for particular bases.

Modulation of tRNA methyltransferase activity by competing enzyme systems

Abstract Fetal organs and tumor tissues exhibit elevated levels of tRNA methyltransferase activity when compared to their normal, adult counterparts. In adult liver, kidney and pancreas this is in part due to the presence of a competing enzyme system, glycine methyltransferase. This enzyme competes for the methyl donor S-adenosylmethionine and is also much less sensitive to product inhibition by S-adenosylhomocysteine than are the tRNA methyltransferases. It is low or absent in fetal liver and a number of hepatomas. In an organ under hormonal control, the uterus, tRNA methyltransferase activity responds to changes in the level of estrogen. Again this seems to be due, in part, to the presence of a competing methyltransferase, catechol O-methyltransferase.

Regulation of the tRNA methyltransferases in normal and neoplastic tissues

Abstract The tRNA methyltransferases are under the control of several regulatory systems. The known mechanisms are hormonal, an unrelated enzyme system glycine methyltransferase, which competes for SAM, and natural inhibitors. The glycine methyltransferase is either absent or its activity is profoundly lowered in embryonic and tumor tissue. Novel tRNAs which are qualitatively different from those in normal tissue have been found in every tumor examined.

5 Enzymic Methylation of Natural Polynucleotides

Publisher Summary Permutations of nucleotides in DNA store the information of amino acid sequence. The retrieval of information is also affected by a universal code of nucleotides in mitochondrial RNA (mRNA). The expression of information is achieved by the nucleotide-composed macromolecules ribosomal RNA (rRNA) and transfer RNA (tRNA). These macromolecules are essentially identical in all species. In search of answers to the questions of species, the identity of these macromolecules, and the way the DNA of an invading parasite is prevented from integrating into the DNA, a species-specific imprint on all of these macromolecules—except mRNA—has been developed. This chapter presents the current knowledge of these species-specific modifying enzymes of nucleic acids. It discusses tRNA methyltransferases—occurrence, purification and properties, substrate specificity, ionic stimulation, and regulation (inhibitors, hormones, bacteriophage induction and infection, nononcogenic and oncogenic virus infection, tumor tissues, and biological significance). For rRNA methyltransferases, the occurrence, isolation, and properties and for DNA methyltransferases, the occurrence and properties of bacteria, eukaryotes, the various regulations, and the biological significance are discussed in the chapter. The bacteriophage T3 infection of E.coli induces the production of an enzyme that destroys S-adenosylmethionine, thus essentially eliminating methylation. Bacteriophage T3 can be inactivated by ultraviolet light, but the S-adenosylmethionine hydrolase is still inducible upon infection. This provides a useful tool for studying the effect of methylation on processes within the infected cell.

Cell transformation by exogenous methylated purines.

Abstract Primary Chinese hamster embryo cell cultures generally yield cell lines with a finite lifetime in culture. However, these cells can be converted to continuous lines by exogenous 1-methylguanine and 7-methylguanine, two normal degradation products of tRNA. Continuous lines have also been obtained by treatment with 3-deazaguanine and the carcinogenic hydrocarbon 3,4-benzpyrene, but not with guanine, 3-methylguanine, or the carcinogen l -ethionine. Continuous lines do arise spontaneously at a low frequency, and lines derived after exposure to modified purines have been analyzed and compared to a spontaneously arising continuous line. The spontaneous Chinese hamster cell line has many characteristics of normal finite lines. These control cells exhibit a diploid karyotype, are not tumorigenic in nude mice, and have normal tRNA methyltransferase, methionine adenosyltransferase, and S-adenosylhomocysteine hydrolase activities. However, as compared to finite Chinese hamster cells, these cells are resistant to infection by the tumor virus SV40. The 1-methylguanine and 7-methylguanine transformed lines which have been analyzed in most detail differ greatly with regard to cell morphology. The former appear to be fibroblasts, while the latter are epithelial-like. The 1-methylguanine treated line exhibits a hyperdiploid karyotype with a modal chromosome number of 23. These cells are tumorigenic in nude mice forming well encapsulated, undifferentiated fibrosarcomas. The 7-methylguanine treated line is hypodiploid with a modal chromosome number of 21. This line is not tumorigenic in nude mice. The transformed cell lines derived by treatment with any of the modified purines or the carcinogen 3,4-benzpyrene all demonstrate elevated tRNA methyltransferase activity as well as elevated S-adenosylhomocysteine hydrolase activity. In addition, methionine adenosyltransferase activity is increased in the 3-deazaguanine and benzpyrene derived lines. Elevated levels of methylated degradation products of tRNA may then aid in maintaining the transformed state.

Interaction of mammalian tRNA methyltransferases with heterologous and homologous tRNA.

The tRNA methyltransferases from normal rat liver and Novikoff hepatoma have been compared with respect to their reaction kinetics using mixed E. coli B tRNA, partially methyl-deficient rat liver tRNA and several purified species of E. coli K-12 MO tRNA. The methyl-deficient rat liver tRNA is a poorer substrate for the enzymes from both sources than is E. coli B tRNA in terms of rate of methylation as well as total acceptance of methyl groups. Affinity constants are generally higher for the methyl-deficient rat liver tRNA than for E. coli B tRNA, with the Novikoff hepatoma enzymes in general having larger affinity constants than have the rat liver enzymes. Maximal velocities for the various base specific enzymes are lower with the methyl-deficient rat liver tRNA in the case of both tissues, with the exception of the 1-methylguanine specific enzymes. These enzymes from either rat liver or Novikoff hepatoma exhibit approximately a 2.4-fold greater maximal velocity with methyl-deficient rat liver tRNA. In the case of the purified bacterial tRNA species, the Novikoff hepatoma enzymes again exhibit larger affinity constants than do the rat liver enzymes, indicating a lowered affinity. The elevated levels of these enzymes in tumor tissue may be present to partially compensate for such lowered affinities.

Cytotoxicity of N6-substituted adenosine analogs to cultured trophoblastic tumor cells☆

Abstract Cultured trophoblastic tumor cells were found to have an elevated adenosine kinase activity relative to levels in normal trophoblasts and those derived from a hydatidiform mole. The effect of N 6 substituted adenosine analogs on the various cell lines was investigated to determine whether the differential enzyme levels might, in turn. result in differential sensitivity of the cell lines to the compounds. When cells were exposed to appropriate concentrations of N 6 -methyladenosine or N 6 , N 6 -dimethyladenosine, it was found that the nucleosides were only cytostatic to non-malignant trophoblastic cells derived from placenta or hydatidiform mole, and cell lines of non-trophoblastic origin. However, the same concentrations of the nucleosides were cytotoxic to the malignant trophoblastic cell lines. The primary effect of exposure of cells to N 6 , N 6 -dimethyladenosine appeared to be inhibition of DNA synthesis. A third adenosine analog, N 6 -( Δ 2 -isopentenyl)aden was toxic to almost all cell lines regardless of origin.

An improved rapid assay for S-adenosyl-L-homocysteine hydrolase.

A coupled enzyme system was devised to assay S-adenosyl-L-homocysteine hydrolase activity spectrophotometrically and to stain the hydrolase selectively in polyacrylamide gels. The assay procedure monitors the formation of uric acid derived from the catabolism of the adenosine moiety of S-adenosylhomocysteine. The staining procedure allows the determination of the molecular weight of the hydrolase when extracts are electrophoresed on polyacrylamide gradient gels and compared to standard of known molecular weight. The specificity of the enzyme for the homocysteine moiety was also investigated by testing modified compounds as substrates. All the analogs tested were inactive as substrates for hydrolysis, indicating a strict specificity.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate elevates serum progesterone levels

Summary The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA), the most potent promoter of two-stage carcinogenesis in mouse skin, simulates some of the early effects of several carcinogens in immature chicks. Actinomycin D, ethionine, thioacetamide (non-mutagenic in the Ames Salmonella test) and kepone are known to increase serum progesterone concentrations and induce synthesis of ovalbumin in chicks. Intraperitoneal administration of TPA to chicks caused a 5- to 7-fold elevation of serum progesterone levels and induced ovalbumin synthesis. Phorbol ester, 4α-phorbol-12,13-didecanoate, which is inactive as a mouse skin tumor promoter, did not increase serum progesterone levels nor did it induce ovalbumin synthesis.

tRNA METHYLTRANSFERASES IN NORMAL AND NEOPLASTIC TISSUES

ABSTRACT: The tRNA methyltransferases are a complex family of enzymes which modify the structure of preformed transfer RNA by the insertion of methyl groups into specific positions in the four major nucleotides comprising tRNA. They are ubiquitously distributed in procaryotes and eucaryotes. The enzymes are species, organ, base, and even site specific for particular bases.