Fan Z, Lu Y, Wu X, DeBlasio A, Koff A, Mendelsohn J
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.
J Cell Biol. 1995 Oct;131(1):235-42. doi: 10.1083/jcb.131.1.235.
Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.
一些携带大量表皮生长因子（EGF）受体的培养人类肿瘤细胞系的增殖，反常地受到纳摩尔浓度EGF的抑制。在本研究中，我们调查了暴露于外源性EGF的A431人鳞状癌细胞生长抑制的生化机制。同时，我们研究了一个选定的亚群A431-F，其对EGF介导的生长抑制具有抗性。当A431和A431-F细胞与20 nM EGF一起培养4小时时，我们观察到细胞周期蛋白依赖性激酶2（CDK2）活性显著降低。在A431细胞进一步持续暴露于EGF后，CDK2活性维持在低水平，并伴有持续的G1期阻滞。相比之下，A431-F细胞中早期降低的CDK2活性和G1期积累只是短暂的。我们发现，在早期时间点（4 - 8小时），EGF在对EGF敏感的A431细胞和对EGF有抗性的A431-F细胞中均诱导p21Cip1/WAF1 mRNA和蛋白表达。但仅在A431细胞中，添加EGF后p21Cip1/WAF1表达在长达5天的时间内持续维持在显著升高的水平。EGF对p21Cip1/WAF1的诱导可被特异性EGF受体酪氨酸激酶抑制剂 tyrphostin AG1478抑制，这表明p21Cip1/WAF1的诱导是EGF激活受体酪氨酸激酶的结果。我们还证明，增加的p21Cip1/WAF1与CDK2和增殖细胞核抗原（PCNA）均相关。综上所述，我们的结果表明p21Cip1/WAF1是EGF诱导A431细胞G1期阻滞和生长抑制的重要介质。
