title: Reversion of transcriptional repression of Sp1 by 5 aza-2' deoxycytidine restores TGF-beta type II receptor expression in the pancreatic cancer cell line MIA PaCa-2.
abstract: The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transforming 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 caused by a deficit of the transcription factor Sp1. Nuclear run-off assays and Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine, resulted in an increase in the 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 cells led to an increase in RII promoter-chloramphenicol acetyltransferase activity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of responsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MIA PaCa-2 Sp1 transfectants) when treated with mithramycin, an inhibitor of Sp1 binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltransferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the Sp1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivation of the RII gene and thus lack of responsiveness to TGF-beta.
authors.affiliation: Department of Surgery, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
authors.forename: K
authors.initials: K
authors.lastname: Venkatasubbarao
authors.forename: S
authors.initials: S
authors.lastname: Ammanamanchi
authors.forename: M G
authors.initials: MG
authors.lastname: Brattain
authors.forename: D
authors.initials: D
authors.lastname: Mimari
authors.forename: J W
authors.initials: JW
authors.lastname: Freeman
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008926:Plicamycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects* / Q000502:physiology; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016329:Sp1 Transcription Factor; D016212:Transforming Growth Factor beta; D000077209:Decitabine; D015254:DNA Modification Methylases; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D001374:Azacitidine; D008926:Plicamycin