5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor α expression

Academic Article


  • Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor α (TGF-α). TGF-α mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-α promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-α promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-α promoter itself does not account for the observed increase in TGF-α mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-α promoter complex was detected in response to 5-azaC treatment. This 5-azaC- induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-α promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5- azaC-induced complex produced a footprint on the TGF-α promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-α expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-α expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.
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    Digital Object Identifier (doi)

    Author List

  • Tae Ho Shin; Paterson AJ; Grant JH; Meluch AA; Kudlow JE
  • Start Page

  • 3998
  • End Page

  • 4006
  • Volume

  • 12
  • Issue

  • 9