Antisense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells.
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.
Ohtsubo
M
M
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Gamou
S
S
Shimizu
N
N
D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D018844:Cyclin-Dependent Kinases