Reversion of transcriptional repression of Sp1 by 5 aza-2 ' deoxycytidine restores TGF-beta type II receptor expression in the pancreatic cancer cellline MIA PaCa-2

The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transformi ng growth factor beta (TGF-beta) because of a lack of expression of the TGF -beta type II receptor (RII). We show that the lack of RII expression is ca used by a deficit of the transcription factor Sp1. Nuclear run-off assays a nd Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl tr ansferase inhibitor, 5-aza-2 ' -deoxycytidine, resulted in an increase in t he rate of Sp1 transcription, in Sp1 protein expression, and in the binding of Sp1 to the RII promoter. Ectopic expression of Sp1 cDNA in MIA PaCa-2 c ells led to an increase in RII promoter-chloramphenicol acetyltransferase a ctivity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of re sponsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MI A PaCa-2 Spl transfectants) when treat with mithramycin, an inhibitor of Sp l binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltra nsferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the S p1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivatio n of the RII gene and thus lack of responsiveness to TGF-beta.