5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression.
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 alpha (TGF-alpha). TGF-alpha 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-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha 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-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha 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-alpha 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-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha 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-alpha 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.
Shin
T H
TH
Department of Cell Biology, University of Alabama, Birmingham 35294.
Paterson
A J
AJ
Grant
J H
JH
Meluch
A A
AA
Kudlow
J E
JE
D001374:Azacitidine / Q000494:pharmacology*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008545:Melanoma; D008745:Methylation; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D016211:Transforming Growth Factor alpha; D004247:DNA; D001374:Azacitidine