Transcriptional activity of human estrogen receptor (hER) gene was modulated by competition with double-stranded PCR-generated DNA fragments (decoys) that contain 5' upstream sequences of the hER gene. Two DNA fragments belonging to the P1 canonical promoter and the P3 distal promoter, 120 and 102 bp in size respectively, were produced by PCR and directly transfected in MCF7 breast cancer cells. After 24 hours transfection, RT-PCR analysis revealed that the 120 bp decoy significantly reduced the expression of the ER gene and estrogen responsive genes (PR and c-myc),whereas the 102 bp decoy increased the ER mRNA level. An ER unrelated PCR product, used as control, had no activity. The biological activity of these ds DNAs was related to their high stability, binding affinities, and lack of cytotoxicity. These findings suggest that such PCR product decoys may be a non-antisense tool to analyze putative regulatory sequences and to study the function of DNA-binding transcription factors.
Modulation of estrogen receptor gene expression in human breast cancer cells: A decoy strategy with specific PCR-generated DNA fragments
PENOLAZZI, Maria Letizia;LAMBERTINI, Elisabetta;AGUIARI, Gianluca;DEL SENNO, Laura;PIVA, Maria Roberta
1998
Abstract
Transcriptional activity of human estrogen receptor (hER) gene was modulated by competition with double-stranded PCR-generated DNA fragments (decoys) that contain 5' upstream sequences of the hER gene. Two DNA fragments belonging to the P1 canonical promoter and the P3 distal promoter, 120 and 102 bp in size respectively, were produced by PCR and directly transfected in MCF7 breast cancer cells. After 24 hours transfection, RT-PCR analysis revealed that the 120 bp decoy significantly reduced the expression of the ER gene and estrogen responsive genes (PR and c-myc),whereas the 102 bp decoy increased the ER mRNA level. An ER unrelated PCR product, used as control, had no activity. The biological activity of these ds DNAs was related to their high stability, binding affinities, and lack of cytotoxicity. These findings suggest that such PCR product decoys may be a non-antisense tool to analyze putative regulatory sequences and to study the function of DNA-binding transcription factors.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
