RESUMO
Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extra cellular matrix, is highly expressed in non-invasive cells but undetectable levels in highly invasive human glioma cells. The mechanisms responsible for its transcriptional regulation are not well elucidated. In this study, we made several deletion constructs from a 3.6 kb genomic fragment from Hs683 cells containing the 5'-flanking region of the TFPI-2 gene, transiently transfected with these constructs into non-invasive (Hs683) and highly invasive (SNB19) human glioma cells, and assessed their expression by using a luciferase reporter gene. Three constructs showed high promoter activity (pTF5, -670 to +1; pTF6, -312 to +1; pTF2, -1511 to +1). Another construct, pTF8 (-81 to +1), showed no activity. PTF9, a variant of pTF5 in which a further 231 bp fragment (-312 to -81) was deleted, from the [-670 to +1] pTF5 region, also showed no promoter activity. Hence, (-312 to -81) this region is essential for the transcription of TFPI-2 in glioma cells. Sequencing of this promoter region revealed that it has a high G+C content, contains potential SP1 and AP1 binding motifs, and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site, although CAAT boxes were found about -3000 bp upstream of the transcription start site. We also found a strong repressor in the region between -927 to -1181, upstream of the major transcriptional initiation site, followed by positive elements or enhancers between -1511 to -1181. These positive elements masked the silencer effect. Finally TFPI-2 was induced in Hs683 cells transfected with the pTF6 construct (-312 to +1) and stimulated with phorbol-12-myristate-13-acetate (PMA). We conclude that the -312 to +1 region is critical for the minimal and inducible regulation of TFPI-2 in non-invasive (Hs683) and highly invasive (SNB19) human glioma cell lines.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Glicoproteínas/biossíntese , Invasividade Neoplásica/genética , Proteínas de Neoplasias/biossíntese , Regiões Promotoras Genéticas/genética , Sequência de Bases , Sítios de Ligação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Glioma/genética , Glioma/metabolismo , Glicoproteínas/genética , Glicoproteínas/fisiologia , Humanos , Luciferases/biossíntese , Luciferases/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
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.