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DNA methyltransferase levels and altered CpG methylation in the total genome and in the GSTP1 gene in human glioma cells transfected with sense and antisense DNA methyltransferase cDNA.

Publication ,  Journal Article
Antoun, G; Baylin, SB; Ali-Osman, F
Published in: J Cell Biochem
April 2000

This study examines the efficacy of using plasmid expression vectors containing sense and antisense DNA MTase cDNA to both up- and downregulate intracellular DNA MTase levels in human glioma cells. The effects of the changes in MTase levels on global genomic DNA methylation and on the methylation status of CpG dinucleotides in the GSTP1 gene were determined in a glioma cell line that overexpresses the GSTP1 gene. In cells transfected with sense DNA MTase cDNA, MTase gene transcripts increased to a maximum of 2. 5-fold at 24 h, while MTase activity increased to a maximum of 3. 6-fold at 48 h. The effects of antisense MTase cDNA transfections were less pronounced, and levels of MTase gene transcripts and enzyme activity in transfectants were decreased to only, approximately, one-half the levels of controls. The alterations in DNA MTase expression were associated with corresponding changes in the level of global DNA methylation and in the methylation of the GSTP1 gene in the cells, however, with no detectable morphological or cytotoxic effects on the cells. No significant changes in GSTP1 gene expression were detected after the transfections, presumably because of the high levels of basal GSTP1 expression in the cells. Consequently, the p16 gene, known to be repressed transcriptionally by DNA methylation, was examined for the functional effects of the altered MTase levels. The results showed a 2-fold decrease in p16 gene transcripts with the sense MTase transfectants, while in the MTase antisense-transfected cells p16 transcript levels increased by 30%. Together, these results demonstrate the feasibility of using both sense and antisense DNA MTase expression vectors to regulate DNA MTase levels in glioma cells and that, over relatively short periods of time, the alterations in MTase activities are not deleterious to the cells. The system provides a model with which the role of DNA methylation in critical genes and DNA sequences can be investigated in glioma cells.

Duke Scholars

Published In

J Cell Biochem

ISSN

0730-2312

Publication Date

April 2000

Volume

77

Issue

3

Start / End Page

372 / 381

Location

United States

Related Subject Headings

  • Up-Regulation
  • Tumor Cells, Cultured
  • Transfection
  • Time Factors
  • Reverse Transcriptase Polymerase Chain Reaction
  • Phenotype
  • Isoenzymes
  • Humans
  • Glutathione Transferase
  • Glutathione S-Transferase pi
 
Journal cover image

Published In

J Cell Biochem

ISSN

0730-2312

Publication Date

April 2000

Volume

77

Issue

3

Start / End Page

372 / 381

Location

United States

Related Subject Headings

  • Up-Regulation
  • Tumor Cells, Cultured
  • Transfection
  • Time Factors
  • Reverse Transcriptase Polymerase Chain Reaction
  • Phenotype
  • Isoenzymes
  • Humans
  • Glutathione Transferase
  • Glutathione S-Transferase pi