RESUMO
Prostate specific membrane antigen (PSMA), also known as folate hydrolase (FOLH1), is a 100 kDa glycoprotein with elevated expression in prostate epithelial tissue. Expression of PSMA is upregulated as prostate tumor grade increases and is found in the vasculature of many tumors, with no presence in benign tissues. Due to the potential of the regulatory elements of the PSMA promoter and enhancer to be used in gene therapy and as biomarkers for prostate cancer under conditions of androgen ablation during treatment, we sequenced and analyzed the ability of 5.5 kb of PSMA promoter/leader region to promote transcription. A recently discovered enhancer, found in the third intron of the PSMA gene, FOLH1, was also studied. The promoter/leader region sequence provided basal expression in transcription assays, while addition of the enhancer activated transcription 41-fold in transient transfections and 144-fold in stable transfections of the LNCaP prostate cell line. This enhancement of transcription was not found in nonprostate cell lines or prostate cell lines that do not express PSMA. An analysis of the ability of androgens to act via the PSMA promoter/leader region and enhancer to activate transcription in transiently transfected LNCaP cells revealed no significant androgen response using the FOLH1 promoter/leader region and a downregulation of 42% with addition of the enhancer. In stably transfected LNCaP cells, the FOLH1 promoter/leader region produced a 21% downregulation in response to androgens, while addition of the enhancer resulted in a 45% downregulation. These results demonstrate significant upregulation of transcription by the PSMA promoter/enhancer, with specificity for the LNCaP prostate cell line, and downregulation of transcription in response to physiological levels of androgen.
Assuntos
Antígenos de Superfície , Carboxipeptidases/genética , Elementos Facilitadores Genéticos/genética , Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutamato Carboxipeptidase II , Células HeLa , Humanos , Masculino , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Antígeno Prostático Específico/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
BACKGROUND: PSMA expression varies among prostate cell lines. We examined the role of CpG methylation and histone deacetylation in PSMA transcriptional repression in prostate cell lines. MATERIALS AND METHODS: The methylation status of a PSMA CpG island was investigated in LNCaP, DU145 and PC3 prostate cell lines. Cells were treated with a demethylating agent and a histone deacetylase inhibitor to determine if PSMA transcription could be activated in nonexpressing cells. A transfection assay with methylated and unmethylated PSMA promoter/enhancer-driven luciferase expression constructs was performed to examine the effect of methylation on transcription. RESULTS: The PSMA CpG island was only methylated in DU145 cells but transcription could not be activated by demethylation or histone deacetylase inhibition. Methylation repressed PSMA transcription in LNCaP cells. CONCLUSION: Although promoter methylation represses PSMA transcription in LNCaP cells, another method inhibits PSMA expression in DU145 and PC3 cells.