5-methyldeoxycytidine and Cell-Transformation--Viral

This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past thirty years. The text does not attempt to give a complete and meticulous account of the many relevant and excellent reports published by many other laboratories, so it is not a review of the field in a conventional sense. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proven their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (i) the de novo methylation of integrated foreign genomes; (ii) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (iii) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (iv) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (v) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; what role has food-ingested DNA played in the elaboration of this mechanism?

Topics: Adenoviridae; Animals; Cell Transformation, Viral; Deoxycytidine; Diet; DNA; DNA Methylation; DNA Modification Methylases; DNA, Viral; Gene Silencing; Genome, Human; Genome, Viral; Genomic Imprinting; Humans; Mice; Mice, Transgenic; Promoter Regions, Genetic; Proto-Oncogenes

In rat kidney cells transformed by avian sarcoma virus (B77 strain) DNA is hypomethylated (2.61 +/- 0.07%) when compared to DNA extracted from normal cells (3.33 +/- 0.11%) as revealed by high-performance liquid chromatography analysis. Kinetics studies showed that no significant differences could be detected between DNA methyltransferase activities from normal and transformed cells with regard to apparent Vmax, apparent Km for S-adenosylmethionine (2.32 X 10(-6) M and 6.64 X 10(-6) M respectively) and apparent Ki for S-adenosylhomocysteine (9.2 X 10(-7) M and 7.8 X 10(-7) M respectively), when unmethylated duplex DNA was used as second substrate. Equivalent ratios of S-adenosylmethionine over S-adenosylhomocysteine were measured in each cell type and DNA methyltransferase activities from both sources were found to be strictly additive. These results show that the hypomethylation of DNA detected in transformed cells is related neither to alterations of enzymatic activities extracted from nuclei nor to unbalanced S-adenosylmethionine/S-adenosylhomocysteine ratios.

Topics: Animals; Avian Sarcoma Viruses; Cell Nucleus; Cell Transformation, Viral; Cells, Cultured; Chromatography, High Pressure Liquid; Deoxycytidine; DNA; DNA (Cytosine-5-)-Methyltransferases; Fibroblasts; Kidney; Kinetics; Rats; S-Adenosylhomocysteine; S-Adenosylmethionine

The incorporation into DNA of 5-bromocytosine and 5-iodocytosine, derived from their respective administered deoxyribonucleoside analogs, has been demonstrated in studies with cells infected with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and in cells transformed with the thymidine kinase gene of HSV-1. No significant incorporation of iodocytosine or iodouracil occurred in the DNA of uninfected or nontransformed cells when the deaminating enzymes were inhibited, in accord with past studies in our laboratory with 5-bromodeoxycytidine and tetrahydrouridine. When 2'-deoxytetrahydrouridine, a potent inhibitor of cytidine deaminase and dCMP deaminase, was utilized, all the counts in DNA that were derived from [(125)I]iododeoxycytidine appeared as iodocytosine in HSV-infected cells. In the absence of a deaminase inhibitor, 32 to 45% of the counts associated with DNA pyrimidines appeared as iodocytosine, and 55 to 68% appeared as iodouracil in HSV-infected cells. Substantial incorporation of iodocytosine (16%) occurred in cells transformed with the HSV thymidine kinase gene, suggesting the importance of the specificity of cellular nucleoside kinases and the activity of the deaminases in presenting unmodified bases to an undiscriminating polymerase. Incorporation into DNA of bromocytosine derived from [(3)H]bromodeoxycytidine was demonstrated in HSV-2 infected cells; very little incorporation of bromocytosine compared with bromouracil could be demonstrated in these cells in the absence of inhibition of the deaminases (19% of the total counts associated with pyrimidines with deaminase inhibition and 1.5% without). Limited studies with 5-methyl[5-(3)H]deoxycytidine indicated essentially no (or very little) incorporation of this analog as such in the DNA of HSV-1- and HSV-2-infected and -transformed cells. This suggests an exclusion or repair mechanism preventing inappropriate methylcytosine incorporation in DNA. The addition of nucleoside and deoxyribonucleoside deaminase inhibitors, which leads to the incorporation of 5-halogenated analogs of deoxycytidine into DNA as such, does not impair their antiviral activity. We infer from studies with 4-N-alkyl (ethyl and isopropyl)-substituted analogs of iododeoxycytidine that they are incorporated as such into DNA without deamination and effectively inhibit the virus at concentrations that are marginally toxic. Among the several reasons presented for the heightened potential efficacy of analogs of deoxycytidine

Topics: Animals; Antiviral Agents; Bromodeoxycytidine; Cell Transformation, Viral; Cytidine Deaminase; Deamination; Deoxycytidine; DNA, Viral; Kinetics; Simplexvirus; Structure-Activity Relationship; Thymidine Kinase; Tritium