Ohtsubo M, Gamou S, Shimizu N
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Oncogene. 1998 Feb 12;16(6):797-802. doi: 10.1038/sj.onc.1201588.
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.
A431细胞过度产生表皮生长因子（EGF）受体并拥有无活性的p53蛋白。有人提出，细胞周期蛋白依赖性激酶（CDK）抑制剂p21/WAF1在EGF诱导的A431细胞周期停滞中起关键作用。在此，我们通过转染与WAF1基因5'区域相对应的18聚体反义寡核苷酸（AS/WAF1），研究了WAF1基因转录在EGF诱导的细胞周期停滞中的作用。当用EGF处理A431细胞时，观察到一系列反应，包括EGF受体酪氨酸残基立即过度磷酸化、WAF1 mRNA和p21/WAF1蛋白积累、作为CDK-细胞周期蛋白底物的RB蛋白去磷酸化以及细胞周期停滞。在存在AS/WAF1的情况下，EGF诱导EGF受体酪氨酸磷酸化，但WAF1 mRNA减少到一半；不再观察到p21/WAF1蛋白积累及其下游反应；A431细胞持续生长。因此，反义转染有效地阻止了A431细胞的EGF诱导停滞。这些观察结果表明，p21/WAF1蛋白是EGF介导的A431细胞周期停滞的主要效应分子。
