A Novel Triplex-Forming Oligonucleotide Targeted to Human Cyclin D1 (bcl-1, Proto-Oncogene) Promoter Inhibits Transcription in HeLa Cells ² Hyung-Gyoon Kim ‡,| and Donald M. Miller* ,‡,§ Department of Biochemistry and Molecular Genetics, and Bolden Laboratory, Department of Medicine, ComprehensiVe Cancer Center, Birmingham, Alabama 35294-0001 ReceiVed September 29, 1997 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. Oligonucleotides can form triple helical structures with polypurine tracts present in duplex DNA in a sequence specific manner. There are two widely known structural motifs by which these triple helical DNA form. Under acidic conditions, pyrimidine rich oligonucleotides bind in the major groove of DNA so that the triplex forming oligonucleotide is parallel to the purine strand of the duplex target. Acidic conditions are required for triplex formation by oligonucle- otides containing cytosine since the cytosine residue must be protonated to form triplex. Sequence specificity is derived from the ability of thymine to recognize adenine‚thymine base pairs (T*A‚T base triplets) and cytosine (C + ) to recognize guanine‚cytosine base pairs (C + *G‚C triplets) (1). Another class of triple helix was described by Cooney et al., who demonstrated that antiparallel purine rich oligo- nucleotides can specifically bind to purine tracts of double helical DNA in the major groove (2). Sequence specificity is based on the fact that guanine recognizes G‚C base pairs (G*G‚C) and adenine recognizes A‚T base pairs (A*A‚T). Although these purine rich oligonucleotides were originally thought to bind to DNA in a parallel orientation, Beal and Dervan showed that third strands bind to their target site in an antiparallel orientation with respect to the purine rich strand of duplex target (3). Sequence specific intermolecular triple helix formation occurs in vitro, which raises the possibility of manipulating gene expression through triple helix formation. Maher et al. utilized triplex formation to inhibit restriction endonu- clease cleavage at sites located in the vicinity of triplex forming regions (4). Triplex formation inhibits binding of the eukaryotic transcription factor Sp1 (5, 6). Cooney et al. have shown that a homopurine oligonucleotide, which forms triple helix within a region of the c-myc P1 promoter, inhibits the in vitro transcription of the c-myc gene (2). The cyclins are a major group of cell cycle regulatory proteins. They were first identified as proteins that ac- cumulate during the cell cycle and are degraded rapidly in mitosis (7-12). The regulated synthesis and degradation of cyclin proteins appear to be critical for proper cell cycle control. Association of cyclins with cyclin-dependent ki- nases results in the subsequent activation of the complex and may activate specific targets whose phosphorylation is important to cell-cycle transition (13, 14). There are five distinct classes of mammalian cyclins termed A-E, and the synthesis and function of these cyclins display cell cycle specificity. Cyclin D1 is encoded by the CCND1 gene located on chromosome 11q13 (15) which has also been known as the PRAD1 proto-oncogene and BCL1 proto- oncogene (16). Cyclin D1 is a nuclear protein whose expression is closely related to the cell cycle transition from G1 to S (17). Since loss of cell cycle control may contribute to tumor formation, it is interesting that cyclin D1 has been ² This work was supported by NIH Grants RO1 CA42664 and RO1 CA54380, Department of Army Grant DAMD 17-93-J-3018, a Veterans Administration Merit Review Grant, and Share Foundation Grants (to D.M.M.). * Author to whom correspondence should be addressed. Tele- phone: (205) 934-1977. Fax: (205) 975-6911. ‡ Department of Biochemistry and Molecular Genetics. § Department of Medicine. | Current address: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL. 2666 Biochemistry 1998, 37, 2666-2672 S0006-2960(97)02399-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/07/1998