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A nonantisense sequence-selective effect of a phosphorothioate oligodeoxynucleotide directed against the epidermal
J M Coulson1, D R Poyner, A Chantry
1Pharmaceutical Sciences Institute, Aston University, Birmingham, UK.
Abstract:
The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line, that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense, mechanism affecting receptor tyrosine kinase activity.
Insights
Antisense oligodeoxynucleotides (ODNs) targeting epidermal growth factor receptor (EGFr) showed promise in reducing carcinoma cell proliferation. The ODN inhibited EGFr tyrosine kinase activity through a nonantisense mechanism, offering a potential new therapeutic strategy.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Epidermal growth factor receptor (EGFr) overexpression is linked to various carcinomas and poor prognosis.
- Targeting EGFr expression is a potential therapeutic strategy for cancer treatment.
Purpose of the Study:
- To design and evaluate antisense oligodeoxynucleotides (ODNs) targeting the EGFr mRNA initiation codon.
- To assess the efficacy of these ODNs in tumor-derived cell lines with amplified EGFr levels.
Main Methods:
- Designed a 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) complementary to the EGFr mRNA initiation codon.
- Tested erbB1AS15 on A431 cells (high EGFr) and control cell lines (low EGFr).
- Utilized Northern and Western blotting to assess EGFr mRNA and protein levels; measured autophosphorylation and tyrosine kinase activity.
Main Results:
- erbB1AS15 significantly reduced A431 cell proliferation and altered cell morphology.
- No reduction in EGFr mRNA or protein levels was observed, indicating a nonantisense mechanism.
- Significant reduction in EGFr autophosphorylation and direct inhibition of EGFr tyrosine kinase activity by erbB1AS15.
Conclusions:
- The PS ODN erbB1AS15 inhibits carcinoma cell proliferation and alters morphology via a sequence-selective, nonantisense mechanism.
- This mechanism involves direct inhibition of EGFr tyrosine kinase activity, not affecting ligand binding or dimerization.
- erbB1AS15 represents a novel therapeutic approach targeting EGFr signaling in cancer.