Increased expression of unmethylated CDKN2D by 5-aza-2'-deoxycytidine in human lung cancer cells

Oncogene. 2001 Nov 22;20(53):7787-96. doi: 10.1038/sj.onc.1204970.


DNA hypermethylation of CpG islands in the promoter region of genes is associated with transcriptional silencing. Treatment with hypo-methylating agents can lead to expression of these silenced genes. However, whether inhibition of DNA methylation influences the expression of unmethylated genes has not been extensively studied. We analysed the methylation status of CDKN2A and CDKN2D in human lung cancer cell lines and demonstrated that the CDKN2A CpG island is methylated, whereas CDKN2D is unmethylated. Treatment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methyltransferase 1, induced a dose and duration dependent increased expression of both p16(INK4a) and p19(INK4d), the products of CDKN2A and CDKN2D, respectively. These data indicate that global DNA demethylation not only influences the expression of methylated genes but also of unmethylated genes. Histone acetylation is linked to methylation induced transcriptional silencing. Depsipeptide, an inhibitor of histone deacetylase, acts synergistically with 5-Aza-CdR in inducing expression of p16(INK4a) and p19(INK4d). However, when cells were treated with higher concentrations of 5-Aza-CdR and depsipeptide, p16(INK4a) expression was decreased together with significant suppression of cell growth. Interestingly, p19(INK4d) expression was enhanced even more by the higher concentrations of 5-Aza-CdR and depsipeptide. Our data suggest that p19(INK4d) plays a distinct role from other INK4 family members in response to the cytotoxicity induced by inhibition of DNA methylation and histone deacetylation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Azacitidine / analogs & derivatives*
  • Azacitidine / pharmacology*
  • Blotting, Southern
  • Blotting, Western
  • Cell Cycle Proteins*
  • Cell Survival / drug effects
  • CpG Islands / genetics
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism
  • Cyclin-Dependent Kinase Inhibitor p19
  • DNA Methylation / drug effects*
  • DNA Modification Methylases / metabolism
  • Decitabine
  • Gene Expression Regulation, Neoplastic / drug effects*
  • Gene Silencing
  • Histone Deacetylase Inhibitors
  • Histone Deacetylases / metabolism
  • Humans
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / metabolism
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Tumor Cells, Cultured


  • CDKN2D protein, human
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p16
  • Cyclin-Dependent Kinase Inhibitor p19
  • Histone Deacetylase Inhibitors
  • RNA, Messenger
  • Decitabine
  • DNA Modification Methylases
  • Histone Deacetylases
  • Azacitidine