A novel triplex-forming oligonucleotide targeted to human cyclin D1 (bcl-1, proto-oncogene) promoter inhibits transcription in HeLa cells.

Abstract

The cyclin D1/bcl-1 proto-oncogene is one of a series of genes encoding proteins which regulate the cell cycle and are involved in the multistep process of tumorigenesis. Translocation of the cyclin D1 proto-oncogene is a common event in B cell lymphoma, and cyclin D1 amplification occurs in breast, esophageal, hepatocellular, and head/neck carcinomas. The human cyclin D1 proto-oncogene promoter contains an 18-base pair purine-pyrimidine rich motif with three C.G interruptions. This motif is a potential target for purine.purine. pyrimidine triplex formation. We have designed a G-rich antiparallel triplex forming oligonucleotide (TFO) targeted to this region. Electrophoretic mobility shift analysis (EMSA) shows that this purine-pyrimidine rich motif is a binding site for the transcription factor Sp1 and that triplex formation by the target sequence prevents the binding of recombinant Sp1. The exact location of triplex formation was confirmed by DNase I footprinting. In an attempt to increase stability, we have used modified phosphorothioate oligonucleotides for cell culture experiments. Triplex formation by the cyclin D1 targeted phosphorothioate oligonucleotide occurs with a binding affinity approximately equal to that of phosphodiester oligonucleotides. This phosphorothioate modified TFO targeted to cyclin D1 also inhibits transcription of the cyclin D1 promoter in HeLa cells, as demonstrated by a decrease in luciferase expression from a stably integrated human cyclin D1 promoter driven luciferase construct. This suggests that triplex formation may represent a gene specific means of inhibiting cyclin D1 expression.