Nomura Masaaki, He Zhiwei, Koyama Ichiko, Ma Wei-Ya, Miyamoto Ken-ichi, Dong Zigang
Department of Hospital Pharmacy, School of Medicine, Kanazawa University, Kanazawa, Japan.
Mol Carcinog. 2003 Sep;38(1):25-32. doi: 10.1002/mc.10140.
Our previous study demonstrated that phosphatidylinositol 3-kinase (PI3K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells.
我们之前的研究表明，磷脂酰肌醇3激酶（PI3K）对于表皮生长因子（EGF）诱导小鼠表皮JB6细胞的细胞转化是必需的。Akt和雷帕霉素的哺乳动物靶点（mTOR）被视为PI3K的下游效应器。因此，在本研究中，我们使用雷帕霉素（一种特异性mTOR抑制剂）和表达Akt1显性负突变体（DNM-Akt1）的细胞，研究了Akt和mTOR在EGF诱导JB6细胞发生细胞转化中的作用。我们发现，用雷帕霉素处理细胞可抑制EGF诱导的细胞转化，但在72小时时仅轻微抑制JB6细胞增殖。虽然PI3K抑制剂LY294002可减弱EGF诱导的活化蛋白1（AP-1）激活，但用雷帕霉素处理并不影响AP-1活性。用雷帕霉素处理可抑制EGF诱导的核糖体p70 S6蛋白激酶（p70 S6K，一种mTOR下游靶点）的磷酸化和激活，但对Akt的磷酸化和激活没有影响。雷帕霉素对EGF诱导的细胞外信号调节蛋白激酶（ERK）的磷酸化也没有影响。我们发现，将DNM-Akt1导入JB6小鼠表皮Cl 41（JB6 Cl 41）细胞可抑制EGF诱导的细胞转化，而不阻断细胞增殖。DNM-Akt1的表达也抑制了EGF诱导的p70 S6K激活以及Akt激活。这些结果表明，Akt/mTOR信号通路参与了EGF诱导的JB6细胞的细胞转化。
