RESUMEN
We have compared the steroid regulation of human genes encoding prostatic acid phosphatase (hPAP), prostate-specific antigen (hPSA), and prostate-specific glandular kallikrein (hK2) at the level of transcription. Reporter constructs of hPAP promoter covering the region -734/+467 were functional in both prostatic (LNCaP and PC-3) and nonprostatic (CV-1) cell lines in transient transfections. hPAP -231/+50 with eight identified transcription factor-binding sites showed the highest, and hPAP -734/+467 showed the lowest transcriptional activity in CV-1 cells. The hPAP promoter could not be induced with androgen, glucocorticoid, or progesterone, contrary to the hPSA (-620/+40) and hK2 (-493/+27) promoters in PC-3 cells cotransfected with the respective steroid receptor expression vector. Therefore, steroids cannot directly regulate hPAP gene expression via receptor binding to steroid response elements at -178 and +336, which have been shown to have androgen receptor-binding ability in vitro. Glucocorticoid was the most powerful activator of the hPSA construct at 10-nM steroid concentrations. On the contrary, glucocorticoid stimulation of the transcriptional activity of the hK2 construct was the weakest among the tested steroids. The results indicate that the steroid response elements in the proximal promoters of hPSA and hK2 genes are not androgen specific, offering the molecular basis for the expression of these genes outside the prostate in tissues containing steroid receptors.
Asunto(s)
Fosfatasa Ácida/genética , Regulación de la Expresión Génica/efectos de los fármacos , Calicreínas/genética , Antígeno Prostático Específico/genética , Próstata/enzimología , Esteroides/farmacología , Andrógenos/farmacología , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Huella de ADN , Glucocorticoides/farmacología , Haplorrinos , Humanos , Masculino , Progesterona/farmacología , Regiones Promotoras Genéticas , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
The transfection of well-differentiated sensitive cells requires careful optimization of the conditions used. In addition to the transfection method chosen, the amount of cells plated, and thus the density of the cells, as well as the influence of the serum concentration play a critical role in the case of LNCaP cells. We also found out that the only appropriate control plasmid for the transfection efficiency was pCMV beta-gal driven by the cytomegalovirus promoter.