Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay.

Abstract

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...

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells

“…Therefore, selective modulation of erbB signaling pathways has emerged as a potential therapeutic target in cancer. Indeed, strategies to inhibit erbB receptors using antagonists (Arteaga et al, 1994;Pietras et al, 1994;Deshane et al, 1996), antisense (Vaughn et al, 1995), or synthetic inhibitors (Fry et al, 1994;Miller et al, 1994;Zhang and Hung, 1996) have proven e cacy in experimental models, and many of these approaches are currently under clinical trials. Immunological approaches using erbB-2 antagonists have shown that erbB signaling regulates a number of mechanisms that can a ect cellular response to chemotherapy, i.e.…”

SupraMolecular BioVectors (SMBV) improve antisense inhibition of erbB-2 expression

“…optimal to obtain maximum cumulative ODN uptake (Berton et al, 1997). According to the supplier's recommendations, a minimal 3-h incubation of DOTAP-ODN complex with cells allows optimal ODN uptake, and lipofection of SK-Br-3 cells has been shown to be optimal after a 4-h incubation with ODN-cationic lipid complex (Vaughn et al, 1995).…”

“…This analysis was performed as described previously (Vaughn et al, 1995). Briefly, 7 x 104 cells were seeded per well in 24-well plates and treated, as described above, in a final volume of 300 pl.…”

“…A challenging approach consists of the use of antisense oligodeoxynucleotides (ODNs), in the inhibition of P185erbB-2 expression, designed to hybridize to erbB-2 mRNA. Such antisense ODNs inhibit P1 85erbB-2 expression in vitro and, in turn, cell proliferation (Bertram et al, 1994;Brysch et al, 1994;Colomer et al, 1994;Vaughn et al, 1995;Wiechen and Dietel, 1995).…”

“…Nevertheless, the use of ODNs is still limited by two major drawbacks: their poor diffusion through plasma membranes and their rapid degradation by nucleases (Helene and Toulme, 1990). The antisense inhibition of PI85ebB-2 in SK-Br-3 cells, a human mammary adenocarcinoma cell model overexpressing the transmembrane receptor, has thus necessitated the use of either native phosphodiester ODNs at a very high concentration (Colomer et al, 1994) or nuclease-resistant phosphorothioate ODNs, carried by commercialized cationic lipids or not (Bertram et al, 1994;Brysch et al, 1994;Vaughn et al, 1995). Because the evidence is becoming stronger for the non-sequencespecific effects of phosphorothioate ODN (Stein and Cheng, 1993;Gura, 1995;Stein, 1995), the development of ODN carriers would seem necessary to improve both the transport and the stability of native ODNs, while preserving their target specificity.…”