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Antisense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells
M Ohtsubo1, S Gamou, N Shimizu
1Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Abstract:
A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5' region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.
Insights
Epidermal Growth Factor (EGF) triggers cell cycle arrest in A431 cells by increasing p21/WAF1 protein. Inhibiting WAF1 gene transcription with antisense oligonucleotides blocked this EGF-induced cell cycle arrest, confirming p21/WAF1
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- A431 cells exhibit hyperactive Epidermal Growth Factor (EGF) receptors and non-functional p53.
- p21/WAF1, a cyclin-dependent kinase (CDK) inhibitor, is implicated in EGF-induced A431 cell cycle arrest.
Purpose of the Study:
- To investigate the specific role of WAF1 gene transcription in EGF-mediated cell cycle arrest in A431 cells.
Main Methods:
- Transfection of A431 cells with an 18-mer antisense oligonucleotide (AS/WAF1) targeting the WAF1 gene's 5' region.
- Treatment of transfected and control cells with EGF.
- Analysis of EGF receptor phosphorylation, WAF1 mRNA and p21/WAF1 protein levels, RB protein dephosphorylation, and cell cycle progression.
Main Results:
- EGF treatment induced EGF receptor hyperphosphorylation, WAF1 mRNA and p21/WAF1 protein accumulation, RB protein dephosphorylation, and cell cycle arrest.
- AS/WAF1 transfection significantly reduced WAF1 mRNA levels by half.
- In the presence of AS/WAF1, EGF-induced p21/WAF1 accumulation and downstream effects were abolished, leading to continuous cell growth.
Conclusions:
- WAF1 gene transcription and subsequent p21/WAF1 protein accumulation are essential for EGF-induced cell cycle arrest in A431 cells.
- p21/WAF1 acts as a major effector molecule in the EGF signaling pathway leading to cell cycle arrest in this cancer cell line.