Growth inhibitory concentrations of EGF induce p21 (WAF1/Cip1) and alter cell cycle control in squamous carcinoma cells.
Growth inhibitory concentrations of EGF induce p21 (WAF1/Cip1) and alter cell cycle control in squamous carcinoma cells.
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作者：
Judit Jakus;W. Yeudall
先前的研究报道了纳摩尔浓度的表皮生长因子（EGF）抑制A431鳞状细胞癌细胞的生长，EGF是一种有效的上皮来源细胞的有丝分裂原。在这项研究中，我们研究了潜在的机制，通过EGF介导的抑制角质形成细胞的生长可能会发生通过分析A431，HN 6和HN 30角质形成细胞的细胞周期调节机制的成分在生长抑制或生长刺激剂量的EGF的存在下。与对照组相比，25 pM EGF处理A431、HN 6和HN 30细胞后，[3 H]胸苷掺入量增加。暴露于2.5 nM EGF减少[3 H]胸苷掺入A431细胞和HN 6细胞的11%和70%的控制水平，分别，而HN 30细胞继续增殖EGF的存在下。[3 H]胸苷掺入试验进行了超过24小时，揭示了在A431细胞的DNA合成的抑制后，12小时暴露于2.5 nM EGF相比，未处理的细胞。流式细胞术研究表明，在加入2.5 nM EGF后，细胞在G 0/G1期积累，但在加入25 pM EGF后，细胞在G 0/G1期积累。Western blot分析显示在生长抑制条件下A431和HN 6细胞中p21（WAF 1/CIP 1/SDI 1）蛋白水平升高。刺激剂量的EGF在这些细胞中不诱导p21。北方印迹杂交显示，在A431细胞暴露于2.5 nM EGF后4 h内，p21 mRNA水平升高，24 h后仍高于基础水平。体外激酶试验表明，CDK 2和CDK 6活性的时间差异与EGF浓度有关。免疫复合物Western印迹显示，2.5 nm EGF处理的A431细胞中p21与CDK 2和CDK 6的相关性增加。此外，观察到PCNA与p21和CDK 6的相关性的时间变化。这些数据表明，p21是一个可能的介质EGF诱导的生长抑制，可能通过涉及隔离的PCNA和抑制CDK活性的机制。
Previous studies have reported inhibition of A431 squamous carcinoma cell growth by nanomolar concentrations of epidermal growth factor (EGF), a potent mitogen for cells of epithelial origin. In this study, we examined potential mechanisms through which inhibition of keratinocyte growth mediated by EGF might occur by analysing components of the cell cycle regulatory machinery in A431, HN6 and HN30 keratinocytes in the presence of growth inhibitory or growth stimulatory doses of EGF. Treatment of cells with 25 pM EGF produced an increase in [3H]thymidine incorporation in A431, HN6 and HN30 cells, with respect to control cultures. Exposure to 2.5 nM EGF reduced [3H]thymidine incorporation in A431 cells and HN6 cells to 11% and 70% of control levels, respectively, whereas HN30 cells continued to proliferate in the presence of EGF. [3H]thymidine incorporation assays carried out over 24 h revealed repression of DNA synthesis in A431 cells after 12 h exposure to 2.5 nM EGF compared to untreated cells. Flow cytometry studies demonstrated accumulation of cells in G0/G1 after addition of 2.5 nM, but not 25 pM EGF. Western blot analysis revealed elevation of p21 (WAF1/CIP1/SDI1) protein levels in A431 and HN6 cells under growth-inhibitory conditions. Stimulatory doses of EGF did not induce p21 in these cells. Northern blot hybridization demonstrated elevated levels of p21 mRNA within 4 h of exposure of A431 cells to 2.5 nM EGF, which remained elevated above basal levels at 24 h. In vitro kinase assays demonstrated temporal differences in CDK2 and CDK6 activities which were related to EGF concentration. Immunocomplex Western blotting demonstrated increased association of p21 with CDK2 and CDK6 in A431 cells treated with 2.5 nm EGF. Furthermore, temporal alterations in the association of PCNA with p21 and with CDK6 were observed. The data indicate that p21 is a likely mediator of EGF-induced growth-inhibition, probably through mechanisms involving sequestration of PCNA and inhibition of CDK activity.