A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements ofantisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%1, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense-effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.Overexpression of the ERBB2 tyrosine kinase receptor is a common feature of human breast cancer (1-3). High level expression is associated with increased transcription, frequently due to gene amplification (4, 5). Both ERBB2 gene amplification and high level expression appear to be early events in the progression of breast cancer, and these properties are maintained during metastatic spread of the disease (6, 7). The oncogenic capacity of ERBB2 can be directly demonstrated by introducing high-level expression vectors into nontransformed fibroblasts, mammary epithelial cells, and transgenic mice, which results in morphologic and growth changes characteristic of neoplasia (8-10). In addition, ERBB2 has a relatively restricted pattern of expression in normal adult tissues (11). These properties make the ERBB2 gene and protein product appealing targets for immune and gene-based therapies for breast cancer.Decreased expression of ERBB2 has been achieved by using monoclonal antibodies specific for the extracellular domain of the protein (12)(13)(14). This has resulted in growth inhibition and, in some cases, differentiation of breast cancer cell lines. Antibodies have been shown to decrease cell surface expression of the ERBB2 receptor via internalization (15). These antibodies may also be acting as receptor agonists, mimicking ligand binding. Ligands for ERBB3 and ERBB4 have similar differentiating effects on breast epithelial cell lines (16)(17)(18)(19)(20).Antibody treatment can be equally effective on cell lines with both normal and high level ERBB2 expression (14), further obscuring th...
2447 is due to a decrease of the rate constant knr in that order (Table IV) and is probably connected to the energetic lowering of the initial state E* of the bridged compounds (compare the red-shifted absorption, Table I) which will decrease &EP. This relative energy lowering of the E* state can also explain the "normal" decreasing lifetime above 77 K before the A* state is reached, as observed for the group 1 derivatives.The state P* is not available for DS-B34 (group 3) and the lifetime and the quantum yield of fluorescence are only weakly affected by the temperature and the solvent polarity. As a matter
The goals of this study were to systematically compare the pharmacokinetics and tissue distribution of phosphorothioate (PS), methylphosphonate (MP), and phosphorodithioate (PS2) oligonucleotide analogs; 15-mers of sequence d-TAC GCC AAC AGC TCC (5'-3') complementary to the AUG region of K-ras were radiolabeled with carbon-14. Oligomers were administered as a single dose in the tail vein of nude mice harboring a K-ras-dependent human pancreatic tumor (CFPAC1). The kinetics of PS, PS2, and MP oligomer availability in the bloodstream was followed. Concentration versus time profiles for all oligomers were biphasic, indicative of a two-compartment model. A rapid distribution phase with t1/2 alpha values of 1 minute or less and an elimination phase with average t1/2 beta values of 24-35 minutes were observed. Volumes of distribution (Vd) were 3.2, 4.8, and 6.3 ml for PS2, MP, and PS, respectively, in comparison to 3.6 ml for sucrose, a fluid-phase marker. Relative tissue drug levels obtained at 1 and 24 hours after administration were kidney > liver > spleen > tumor > muscle. Total kidney and liver oligonucleotide accumulation was approximately 7%-15% of the initial dose, with tumor accumulating 2%-3%. Intact compound was recovered from all tissues, including tumor, as assessed by high-pressure reversed-phase HPLC coupled to radiometric detection. Integrity of the oligonucleotides ranged from 73% in blood to 43%-46% in kidney and liver. Kidney and liver appear to be the primary sites of metabolism. These results demonstrate widespread tissue availability of these compounds and suggest their development as potential antitumor agents.
Antisense activity against erbB-2 of a variety of sulfur-modified oligonucleotides was examined in a breast cancer cell line which overexpresses this oncogene. Using a 15 base anti-erbB-2 sequence previously shown to be effective, various backbone configurations containing phosphoromonothioate or phosphorodithioate linkages were evaluated for antisense activity by a two-color flow cytometric assay. This sequence was effective in inhibiting the production of erbB-2 protein when it was configured as a monothioate at each linkage and as an alternating dithioate/phosphodiester. Both of these compounds were also able to specifically inhibit erbB-2 mRNA expression, indicative of RNase H-mediated activity. The same sequence protected by either three dithioate or three monothioate linkages at each end was ineffective as an antisense reagent, suggesting that endonuclease activity is a significant determinant of the stability of oligonucleotides. Finally, the erbB-2 sequence target was shifted in an effort to improve antisense activity. A new lead sequence was identified that was significantly more effective in inhibiting erbB-2 protein levels and retained activity at lower concentrations.
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