Grana Theresa M, Rusyn Elena V, Zhou Hong, Sartor Carolyn I, Cox Adrienne D
Department of Radiation Oncology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Cancer Res. 2002 Jul 15;62(14):4142-50.
Cells transformed by the oncogenic small GTPase, Ras, display a radioresistant phenotype in response to ionizing radiation (IR). To determine the mechanisms by which Ras mediates radioresistance in epithelial cells, we assessed the importance of three major survival pathways that can be activated by Ras [phosphatidylinositol 3-kinase (PI3-K)>Akt, nuclear factor kappaB (NF-kappaB), and Raf>mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)>extracellular signal-regulated kinase] as necessary or sufficient for Ras-mediated radioresistance in matched pairs of RIE-1 rat intestinal epithelial cells expressing oncogenic Ras or empty vector (RIE-Ras and RIE-vector). Inhibiting PI3-K with LY294002 sensitized RIE-1 cells to IR in a dose-dependent manner, indicating that PI3-K is necessary for radioresistance, whereas inhibition of NF-kappaB with the super-repressor IkappaBalpha had little effect on survival. Expression of either the constitutively active catalytic subunit of PI3-K, p110alpha-CAAX, or the Ras effector domain mutant 12V/40C, which retains binding to PI3-K but is impaired in binding to other Ras effectors, was sufficient to confer partial radioresistance. Expression of either a constitutively active form of the serine/threonine kinase Raf-1 or the Ras effector domain mutant 12V/35S, which retains binding to Raf but is impaired in binding to other Ras effectors, was also sufficient to confer partial radioresistance. Surprisingly, however, even complete inhibition of MEK activity by using U0126 resulted in no change in post-IR survival whatsoever. Thus, whereas Raf contributes to Ras-mediated radioresistance, this is accomplished through a MEK-independent pathway. Taken together, these results indicate that multiple pathways, including both PI3-K-dependent and Raf-dependent but MEK-independent signaling, are required for Ras-mediated radioresistance in epithelial cells. Finally, we demonstrate that Ras-mediated radioresistance can be uncoupled from Ras-mediated transformation, in that PI3-K is required for radioresistance but not transformation, whereas MEK and NF-kappaB are required for transformation but not radioresistance in RIE-1 epithelial cells.
由致癌性小GTP酶Ras转化的细胞，在受到电离辐射（IR）时表现出抗辐射表型。为了确定Ras介导上皮细胞抗辐射的机制，我们评估了Ras可激活的三条主要存活途径的重要性[磷脂酰肌醇3激酶（PI3-K）>Akt、核因子κB（NF-κB）以及Raf>丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（MEK）>细胞外信号调节激酶]，它们对于表达致癌性Ras或空载体的配对RIE-1大鼠肠上皮细胞（RIE-Ras和RIE-载体）中Ras介导的抗辐射是否必要或充分。用LY294002抑制PI3-K以剂量依赖的方式使RIE-1细胞对IR敏感，表明PI3-K对于抗辐射是必要的，而用超抑制因子IkappaBalpha抑制NF-κB对细胞存活影响很小。组成型活性PI3-K催化亚基p110alpha-CAAX或Ras效应结构域突变体12V/40C的表达足以赋予部分抗辐射能力，其中12V/40C保留了与PI3-K的结合但与其他Ras效应器的结合受损。组成型活性丝氨酸/苏氨酸激酶Raf-1或Ras效应结构域突变体12V/35S的表达也足以赋予部分抗辐射能力，其中12V/35S保留了与Raf的结合但与其他Ras效应器的结合受损。然而，令人惊讶的是，即使使用U0126完全抑制MEK活性，IR后的细胞存活也没有任何变化。因此，虽然Raf有助于Ras介导的抗辐射，但这是通过一条不依赖MEK的途径实现的。综上所述，这些结果表明，上皮细胞中Ras介导的抗辐射需要多种途径，包括依赖PI3-K和依赖Raf但不依赖MEK的信号传导。最后，我们证明Ras介导的抗辐射可以与Ras介导的转化解偶联，因为PI3-K是抗辐射所必需的，但不是转化所必需的，而MEK和NF-κB是RIE-1上皮细胞转化所必需的，但不是抗辐射所必需的。
