Thimmaiah Kuntebommanahalli N, Easton John, Huang Shile, Veverka Karen A, Germain Glen S, Harwood Franklin C, Houghton Peter J
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA.
Cancer Res. 2003 Jan 15;63(2):364-74.
The mTOR inhibitor rapamycin induces G1 cell cycle accumulation and p53-independent apoptosis of the human rhabdomyosarcoma cell line Rh1. Insulin-like growth factor I (IGF-I) and insulin, but not epidermal growth factor or platelet-derived growth factor, completely prevented apoptosis of this cell line. Because the Ras-Erk1-Erk2 and phosphatidylinositol 3'-kinase (PI3K)-Akt pathways are implicated in the survival of various cancer cells, we determined whether protection from rapamycin-induced apoptosis by IGF-I requires one or both of these pathways. Despite the blocking of Ras-Erk signaling by the addition of PD 98059 (a MEK1 inhibitor) or by the overexpression of dominant-negative RasN17, IGF-I completely prevented rapamycin-induced death. Inhibition of Ras signaling did not prevent Akt activation by IGF-I. To determine the role of the PI3K-Akt pathway in rescuing cells from apoptosis caused by rapamycin, cells expressing dominant-negative Akt were tested. This mutant protein inhibited IGF-I-induced phosphorylation of Akt and blocked phosphorylation of glycogen synthase kinase 3. The prevention of rapamycin-induced apoptosis by IGF-I was not inhibited by expression of dominant-negative Akt either alone or under conditions in which LY 294002 inhibited PI3K signaling. Furthermore, IGF-I prevented rapamycin-induced apoptosis when the Ras-Erk1-Erk2 and PI3K-Akt pathways were blocked simultaneously. Similar experiments in a second rhabdomyosarcoma cell line, Rh30, using pharmacological inhibitors of PI3K or MEK1, alone or in combination, failed to block IGF-I rescue from rapamycin-induced apoptosis. Therefore, we conclude that a novel pathway(s) is responsible for the IGF-I-mediated protection against rapamycin-induced apoptosis in these rhabdomyosarcoma cells.
mTOR抑制剂雷帕霉素可诱导人横纹肌肉瘤细胞系Rh1的G1期细胞周期停滞和不依赖p53的凋亡。胰岛素样生长因子I（IGF-I）和胰岛素可完全阻止该细胞系的凋亡，而表皮生长因子或血小板衍生生长因子则不能。由于Ras-Erk1-Erk2和磷脂酰肌醇3'-激酶（PI3K）-Akt信号通路与多种癌细胞的存活有关，我们确定IGF-I对雷帕霉素诱导的凋亡的保护作用是否需要这两条信号通路中的一条或两条。尽管添加PD 98059（一种MEK1抑制剂）或过表达显性负性RasN17可阻断Ras-Erk信号传导，但IGF-I仍能完全阻止雷帕霉素诱导的细胞死亡。Ras信号的抑制并未阻止IGF-I对Akt的激活。为了确定PI3K-Akt信号通路在挽救细胞免受雷帕霉素诱导的凋亡中的作用，我们检测了表达显性负性Akt的细胞。这种突变蛋白抑制了IGF-I诱导的Akt磷酸化，并阻断了糖原合酶激酶3的磷酸化。单独表达显性负性Akt或在LY 294002抑制PI3K信号传导的条件下，IGF-I对雷帕霉素诱导的凋亡的预防作用均未受到抑制。此外，当Ras-Erk1-Erk2和PI3K-Akt信号通路同时被阻断时，IGF-I仍能阻止雷帕霉素诱导的凋亡。在第二个横纹肌肉瘤细胞系Rh30中，使用PI3K或MEK1的药理学抑制剂单独或联合进行的类似实验，均未能阻断IGF-I对雷帕霉素诱导的凋亡的挽救作用。因此，我们得出结论，一条新的信号通路负责IGF-I介导的对这些横纹肌肉瘤细胞中雷帕霉素诱导的凋亡的保护作用。
