Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents.

Abnormal cell signal transduction arising from protein tyrosine kinases has been implicated in the initiation and progression of a variety of human cancers. Over the past 2 decades pharmaceutical and university laboratories have been involved in a tremendous effort to develop compounds that can selectively modulate these abnormal signalling pathways. Targeting receptor tyrosine kinases, especially the epidermal growth factor receptor subfamily, has been at the forefront of this effort as a result of strong clinical data correlating over-expression of these receptors with more aggressive cancers. There are a variety of strategies under development for inhibiting the kinase activity of these receptors, targeting both the extracellular and intracellular domains. Antibody-based approaches, immunotoxins and ligand-binding cytotoxic agents use the extracellular domain for targeted tumour therapy. Small molecule inhibitors target the intracellular catalytic region by interfering with ATP binding, while nonphosphorylatable peptides are aimed at the intracellular substrate binding region. Compounds that inhibit subsequent downstream signals from the receptor by interrupting intracellular protein recognition sequences are also being investigated. In the past 5 years enormous progress has been made in developing tyrosine kinase inhibitor compounds with sufficient potency, bioavailability and selectivity against this subfamily of receptor tyrosine kinases. The anti-HER2 monoclonal antibody, trastuzumab, for patients with metastatic breast cancer is the first of these inhibitor compounds to gain FDA approval. However, preclinical and clinical trials are ongoing with a variety of other monoclonal antibodies, immunotoxins, and small molecule quinazoline and pyrimidine-based inhibitors. Although their cytotoxic and cytostatic potential has been proven, they are not likely to replace standard chemotherapy regimens as single-agent, first-line therapeutics. Instead, their promising additive and synergistic antitumour effects in combination with standard chemotherapeutics suggest that these novel agents will find their greatest utility and efficacy in conjunction with existing anticancer agents.

Evidence of post-transcriptional regulation of U6 small nuclear RNA.

Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized normal and malignant human cell lines. Transfection and expression of a modified U6 gene containing native promoter, capping, and termination sequences but lacking all highly conserved internal spliceosome sequences produced dose-dependent effects on endogenous U6 gene expression. At low transfection doses, no significant changes in endogenous U6 RNA levels or half-life were noted. However, as the dose of the transfected gene and its expression increased, native U6 RNA levels dramatically decreased in association with an apparent decrease in U6 RNA half-life. Down-regulation of native U6 RNA levels was transient, with recovery noted within 48-96 h in conjunction with declining expression of the ectopic gene. These modulatory effects appeared specific to endogenous U6 transcripts, because no changes were noted in 7sk, U1, U3, or 5S RNA levels or half-lives. Transfection with an unmodified U6 gene did not alter total U6 transcript levels but did produce a similar dose-dependent decrease in U6 RNA half-life. These studies suggest a hitherto unrecognized U6-specific intracellular regulatory mechanism, through which over-accumulation of U6 small nuclear RNA is prevented.

Triplex formation with alpha anomers of purine-rich and pyrimidine-rich oligodeoxynucleotides.

Nuclease-resistant alpha anomers of pyrimidine-rich CT- and purine-rich GA- and GT-containing oligonucleotides were investigated for their triplex-forming potential and compared with their corresponding nuclease-sensitive beta anomers. Both 23mer CT-alpha and 23mer CT-beta had quite similar triplex binding affinities. Synthetic 23mer GT-alpha oligonucleotides were capable of triplex formation with binding affinities slightly lower than corresponding 23mer GT-beta oligonucleotides. The orientation of third strand GT-alpha binding was parallel to the purine strand of the duplex DNA target, whereas the orientation of third strand GT-beta binding was found to be antiparallel. Triplex formation with both GT oligonucleotides showed the typical dependence on magnesium and temperature. In contrast, 23mer GA-alpha oligonucleotides did not support triplex formation in either orientation under a variety of experimental conditions, whereas the corresponding 23mer GA-beta oligonucleotides demonstrated strong triplex formation in the antiparallel orientation. GA-alpha oligonucleotides covalently conjugated to acridine were similarly unable to demonstrate triplex formation. GA-alpha oligonucleotides, in contrast to GT-alpha oligonucleotides, were capable of self-association, detectable by gel retardation and UV spectroscopy, but competing self-association could not fully account for the lack of triplex formation. Thus for in vivo triplex gene regulation strategies using GT oligonucleotides the non-natural alpha anomer may be a feasible alternative to the natural beta anomer, allowing for a comparable degree of triplex formation without rapid cellular degradation. However, alpha anomeric inversion does not appear to be a feasible alternative in applications involving GA oligonucleotides.

Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats.

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.

Inhibition of transcription factor binding to the HER2 promoter by triplex-forming oligodeoxyribonucleotides.

We have identified a 28-bp homopurine/homopyrimidine sequence capable of triple helix (triplex) formation with G+T-rich oligodeoxyribonucleotides (oligos) within the critical proximal promoter of the HER2/neu/c-erbB2 (HER2) proto-oncogene. To investigate the possible therapeutic potential of triplex-forming oligos in HER2 overexpressing breast cancers, we have studied the ability of triplex formation to compete with and to inhibit the binding of a transcription factor to its consensus sequence at an adjacent site. Competition binding assays demonstrate that a triplex-forming oligo can inhibit transcription factor binding in a sequence-specific manner. Moreover, we find that the addition of both nucleotide and non-nucleotide 'tails' to triplex-forming oligos do not confer any enhancement of binding affinity, but provide additional inhibition of transcription factor binding, potentially by steric hindrance.

In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation.

Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of gene-specific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing large quantities of short, sequence-specific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gamma-monomethyl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (and/or ribozymes) for gene regulation, as well as a cost-effective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences.

Detection of triplex-forming RNA oligonucleotides by triplex blotting.

Triplex formation with RNA oligonucleotides and double-stranded (ds) DNA may provide a means of controlling gene expression from specific promoters and/or creating more selective DNA cleaving agents. We report the development of a novel technique, called triplex blotting, designed to detect RNA species capable of triplex formation with radiolabeled dsDNA probes within a background of total cellular RNA. Triplex blotting offers a new approach for screening potential RNA sequences for triplex formation with dsDNA targets, for comparing relative binding affinities of various triplex-forming RNAs and for confirming the specificity of triplex formation of a DNA target probe within total cellular RNA. In addition, the technique allows for repeated probing of the same filter while varying critical hybridization conditions such as pH, temperature or ionic strength.

Characteristics of oligonucleotide uptake in human keratinocyte cultures.

Oligodeoxyribonucleotides have the potential to interfere selectively with cellular protein synthesis by sequence-specific hybridization to DNA or RNA molecules. We have investigated the properties of uptake and intracellular localization of fluorescently labeled oligonucleotides in cultured human keratinocytes using confocal laser scanning microscopy. Unlike many other cell types studied, keratinocytes can internalize oligonucleotides without apparent sequestration in endosomes or cell surface accumulation. Uptake is primarily nuclear and unaltered by sodium azide, monensin, or chloroquine pretreatment. We have verified our results with two different fluorophores, fluorescein and Bodipy, and found similar uptake and distribution patterns in both live and fixed cell populations. Surprisingly, we have found uptake to be heterogeneous within a population, with 15-30% of cells internalizing the oligonucleotides. This percentage is drastically increased to roughly 80% at cell population margins, and after release from M phase arrest. These results on uptake and intracellular localization suggest that keratinocytes may have increased sensitivity as target cells for oligonucleotide based gene regulation strategies.

Characterization and minimization of cellular autofluorescence in the study of oligonucleotide uptake using confocal microscopy.

Intrinsic autofluorescent signals can interfere with extrinsic fluorophore signals when living cells are viewed under a confocal laser scanning microscope. The general pattern of this endogenous fluorescence is initially diffuse and cytoplasmic, but it can redistribute and intensify to become punctate and perinuclear as cells age. To reduce the contribution of autofluorescence when tracking the location of an extrinsic fluorophore, such as a fluorescently-labeled oligonucleotide, laser power settings, aperture settings, laser scanning rates, pH buffering environments, and excitatory wavelengths can be modulated. Decreasing laser power settings and aperture sizes, increasing laser scanning rates and excitatory wavelengths, and surrounding cells in a pH buffer all act to delay the signal transformation. In addition, the presence of an exogenous fluorophore can hasten the autofluorescent redistribution and intensification when compared to similar untreated cells.