Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif.

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine-homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.

Inhibition of transcription elongation in the HER-2/neu coding sequence by triplex-directed covalent modification of the template strand.

Triplex formation may be of potential utility to inhibit the expression of individual genes. We describe the formation of a triple helix in the coding sequence of the HER-2/neu gene. In vitro transcription analysis in the presence and absence of triplex formation demonstrates that an unmodified DNA triplex-forming oligonucleotide is incapable of inhibiting RNA polymerase elongation. Triplex formation by an oligonucleotide-psoralen conjugate was used to form a covalent photoadduct with a thymine on the nontemplate strand of the HER-2/neu gene. In the native HER-2/neu gene, covalent attachment of the triplex-forming oligonucleotide to the nontemplate strand did not prevent RNA polymerase elongation. Using HER-2/neu point mutants that would place the target thymine on the template strand, we demonstrated that covalent modification of the template strand was necessary to inhibit RNA polymerase elongation. Based on these data, we synthesized oligonucleotide-alkylator conjugates that would react with a specific guanine residue on the template strand of the HER-2/neu coding sequence. The oligonucleotide-alkylator conjugates inhibited transcription elongation by T7 RNA polymerase and eukaryotic RNA polymerase II from a HeLa nuclear extract. These studies demonstrate the successful application of triplex-forming oligonucleotide-alkylator conjugates to inhibit transcription elongation in the HER-2/neu gene, and show that covalent modification of the DNA strand used as the transcription template is necessary to prevent RNA polymerase elongation.

The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

Efficient delivery of triplex forming oligonucleotides to tumor cells by adenovirus-polylysine complexes.

Oligonucleotides (ODNs) show great promise in their ability to specifically inhibit single gene expression but must cross the cell membrane, escape the endosomal vesicle, and possibly traverse the nuclear membrane to arrive at their intracellular target molecules. In an attempt to improve the delivery of phosphodiester triplex forming ODNs to malignant cells, we have constructed adenovirus-polylysine (AdpL)-ODN complexes designed to take advantage of the receptor mediated endocytosis of adenoviruses to transfer the ODNs to the cell nucleus. Treatment of several different types of tumor cells in culture by AdpL-ODN complex resulted in superior uptake and persistence of the ODNs compared to both free ODN and cationic lipid-ODN complexes. Nuclear uptake peaks at 4 h and intact ODN persists in the nucleus with a half-life of 12 h. ODN concentrations of 20-70 microM are achieved at 24 h in all monolayer cell lines evaluated to date. ODNs are detected in 50-100% of the total cell population by immunohistochemistry with apparent uptake into vesicles and nuclear localization. Luciferase expression of a co-delivered reporter plasmid suggests that these ODNs are free in the nucleus. AdpL-ODN complexes will provide a valuable tool for delivering unmodified ODNs to the nucleus of malignant cells.

Rhabdoid tumor of the kidney in an adult: review of the literature and report of a case responding to interleukin-2.

Rhabdoid tumor of the kidney is an uncommon and highly aggressive malignancy usually found in the pediatric age group. This tumor does not respond well to aggressive chemotherapy regimens and survival tends to be short. Only two cases of rhabdoid tumor of the kidney occurring in adults have been described previously. The third case of a rhabdoid tumor of the kidney in an adult is presented here. This clinical case report is unique not only because of the rare occurrence of this tumor in adulthood but because the patient described had an objective antitumor response to outpatient low dose interleukin-2. This single case report may have therapeutic implications for other patients with this tumor.

Triplex formation inhibits HER-2/neu transcription in vitro.

Triplex-forming oligonucleotides (TFOs) have been shown to bind to target DNA sequences in several human gene promoters such as the c-myc oncogene, the epidermal growth factor receptor, and the dihydrofolate reductase genes. TFOs have been shown to inhibit transcription in vitro and gene expression in cell culture of the c-myc and other genes. The HER-2/neu oncogene, which is overexpressed in breast cancer and other human malignancies, contains a purine-rich sequence in its promoter, which is favorable for purine:purine:pyrimidine (R:R:Y) triplex formation. Although its function in the HER-2/neu promoter is unknown, this purine-rich site is homologous to a protein-binding sequence in the promoter of the epidermal growth factor receptor that is necessary for efficient transcription of this gene. We have shown that this sequence is a site for nuclear protein binding by incubation with a crude nuclear extract. We describe the formation of an interstrand triplex using a purine-rich oligonucleotide antiparallel to this purine-rich target sequence of the HER-2/neu promoter. Triplex formation by the oligonucleotide prevents protein binding to the target site in the HER-2/neu promoter in vitro. We have shown that this oligonucleotide is a potent and specific inhibitor of HER-2/neu transcription in an in vitro assay. The triplex target site contains a single pyrimidine base that does not conform to the R:R:Y triplex motif. In an attempt to abrogate the potentially destabilizing effects of this pyrimidine base on triplex formation, we have substituted an abasic linker for the pyrimidine residue in the triplex forming oligonucleotide. Triplex formation with the modified oligonucleotide appears to occur with approximately equivalent binding affinity. Triplex formation in the HER-2/neu oncogene promoter prevents transcription in vitro and may represent a future modality for specific inhibition of this gene in vivo.