A Novel Triplex-Forming Oligonucleotide Targeted to Human Cyclin D1 (bcl-1, Proto-Oncogene) Promoter Inhibits Transcription in HeLa Cells

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• 作者：Hyung-Gyoon Kim and Donald M. Miller
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：February 24, 1998
• 年：1998
• 卷：37
• 期：8
• 页码：2666 - 2672
• 全文大小：106K
• 年卷期：v.37,no.8(February 24, 1998)
• ISSN：1520-4995

文摘

The cyclin D1/bcl-1proto-oncogene is one of a series of genes encoding proteins whichregulatethe cell cycle and are involved in the multistep process oftumorigenesis. Translocation of the cyclin D1proto-oncogene is a common event in B cell lymphoma, and cyclin D1amplification occurs in breast,esophageal, hepatocellular, and head/neck carcinomas. The humancyclin D1 proto-oncogene promotercontains an 18-base pair purine-pyrimidine rich motif with three C·Ginterruptions. This motif is a potentialtarget for purine·purine·pyrimidine triplex formation.We have designed a G-rich antiparallel triplexforming oligonucleotide (TFO) targeted to this region.Electrophoretic mobility shift analysis (EMSA)shows that this purine-pyrimidine rich motif is a binding site for thetranscription factor Sp1 and thattriplex formation by the target sequence prevents the binding ofrecombinant Sp1. The exact location oftriplex formation was confirmed by DNase I footprinting. In anattempt to increase stability, we haveused modified phosphorothioate oligonucleotides for cell cultureexperiments. Triplex formation by thecyclin D1 targeted phosphorothioate oligonucleotide occurs with abinding affinity approximately equalto that of phosphodiester oligonucleotides. This phosphorothioatemodified TFO targeted to cyclin D1also inhibits transcription of the cyclin D1 promoter in HeLa cells, asdemonstrated by a decrease inluciferase expression from a stably integrated human cyclin D1 promoterdriven luciferase construct.This suggests that triplex formation may represent a gene specificmeans of inhibiting cyclin D1 expression.