Lack of a role of DNA methylation in tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced synthesis of ornithine decarboxylase messenger RNA in T24 cells.
Association of alteration in DNA methylation pattern in triggering 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transcription of ornithine decarboxylase (ODC) gene in T24 cells was determined. In accord with our previous findings (Archiv. Biochem. Biophys., 262: 326-336, 1988), TPA treatment of T24 cells, cultured in serum-free medium, resulted in a dramatic (approximately 15-fold) increase in ODC activity which was accompanied by a proportional increase in hybridizable amount of ODC mRNA. Data from nuclear run-off transcription assay revealed that TPA-induced accumulation of ODC mRNA is the result of increased transcription initiation. Since DNA hypomethylation has been proposed to be a mechanism involved in the regulation of transcription of some gene(s), we examined the changes in the methylation patterns in the ODC gene isolated from the vehicle (ethanol)- and TPA-treated T24 cells. The autoradiograms resulting from the Southern blot analysis of DNA cleaved with several methylation-sensitive restriction endonucleases [e.g., HpaII, MspI, cfoI (HhaI), SalI, XhoI] exhibited no difference in methylation pattern of ODC gene in T24 cells. Also, a single or chronic application of TPA to either noninitiated or 7,12-dimethylbenz(a)anthracene-initiated mouse skin failed to alter DNA methylation pattern of ODC gene. Furthermore, the hypomethylation agent 5-azacytidine failed to induce ODC mRNA in T24 cells. These results indicate that TPA does not affect the methylation status of ODC gene and hypomethylation may not be sufficient for TPA-increased ODC gene transcription in T24 cells.
Hsieh
J T
JT
Department of Human Oncology, University of Wisconsin, Madison 53792.
Verma
A K
AK
D004247:DNA / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D009955:Ornithine Decarboxylase / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D004247:DNA; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate