title: Methylation of TFPI-2 gene is not the sole cause of its silencing.
abstract: TFPI-2 (tissue factor pathway inhibitor-2) is a serine protease inhibitor that may suppress tumor cell invasion and metastasis. TFPI-2 expression is often lost in cells derived from tumors of diverse organs. We have examined whether aberrant hypermethylation of the 5' end of the TFPI-2 gene is associated with its loss of expression. After 5-azacytidine treatment of three cell lines lacking TFPI-2 expression (HT1080 fibrosarcoma cells, MCF-7 breast carcinoma cells, and LNCaP prostate carcinoma cells), TFPI-2 transcripts could be detected by RT-PCR. In these three cell lines, methylation of the 5' end of the TFPI-2 gene was detected, while two prostate carcinoma cell lines in which the TFPI-2 gene was expressed, PC-3 and DU-145, showed no methylation. However, all the three cell lines which lacked TFPI-2 expression also contained unmethylated TFPI-2 alleles. Furthermore, a transiently transfected TFPI-2 promoter was non-functional in the three cell lines, but function was attained following treatment with 5-azacytidine. Our results indicate that while methylation of the TFPI-2 gene is associated with its silencing, it is not the sole cause, and we suggest that one or more components of pathways regulating TFPI-2 expression have also undergone methylation-associated silencing in these cell lines.
authors.affiliation: Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. nageswararao.chilukuri@na.amedd.army.mil
authors.forename: Chilukuri N
authors.initials: CN
authors.lastname: Rao
authors.forename: Takehiko
authors.initials: T
authors.lastname: Segawa
authors.forename: Jason R
authors.initials: JR
authors.lastname: Navari
authors.forename: Linda
authors.initials: L
authors.lastname: Xu
authors.forename: Shiv
authors.initials: S
authors.lastname: Srivastava
authors.forename: Judd W
authors.initials: JW
authors.lastname: Moul
authors.forename: Benette
authors.initials: B
authors.lastname: Phillips
mesh_terms: D000483:Alleles; D001374:Azacitidine / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D019175:DNA Methylation; D005260:Female; D020868:Gene Silencing; D017930:Genes, Reporter; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008297:Male; D008957:Models, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites / Q000494:pharmacology; D014162:Transfection
chemical_list: D006023:Glycoproteins; D013447:Sulfites; C021240:tissue-factor-pathway inhibitor 2; D008156:Luciferases; D001374:Azacitidine; C009279:sodium bisulfite