Dent P, Reardon D B, Park J S, Bowers G, Logsdon C, Valerie K, Schmidt-Ullrich R
Department of Radiation Oncology, Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Mol Biol Cell. 1999 Aug;10(8):2493-506. doi: 10.1091/mbc.10.8.2493.
Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.
将A431鳞状细胞和MDA - MB - 231乳腺癌细胞暴露于电离辐射下，已发现与表皮生长因子受体（EGFR）酪氨酸磷酸化的短暂增加以及丝裂原活化蛋白激酶（MAPK）和c - Jun氨基末端激酶（JNK）信号通路的激活有关。对A431和MDA - MB - 231细胞进行2 Gy的照射，导致EGFR以及MAPK和JNK信号通路立即出现初级激活（0 - 10分钟），令人惊讶的是，随后还出现了后期延长的次级激活（90 - 240分钟）。通过分子抑制EGFR功能可消除EGFR的初级和次级激活。对EGFR或Ras功能进行分子抑制可消除MAPK信号通路的初级和次级激活。相比之下，分子抑制EGFR功能可消除JNK信号通路的次级激活，但不能消除其初级激活。使用中和单克隆抗体抑制肿瘤坏死因子α受体功能可减弱JNK信号通路 的初级激活。添加针对转化生长因子α（TGFα）的中和单克隆抗体，对照射后EGFR或MAPK和JNK信号通路的初级激活没有影响，但可消除EGFR、MAPK和JNK的次级激活。照射细胞后120 - 180分钟，前体TGFα的裂解增加。与辐射诱导可溶性因子释放一致，照射后120分钟，将照射细胞的培养基转移至未照射细胞中，可诱导EGFR以及MAPK和JNK信号通路的激活。当培养基与针对TGFα的中和抗体孵育时，转移培养基诱导MAPK和JNK激活的能力被阻断。因此，辐射可导致自分泌调节的癌细胞中EGFR以及MAPK和JNK信号通路的初级和次级激活。EGFR以及MAPK和JNK信号通路的次级激活依赖于辐射诱导的TGFα裂解和自分泌作用。在凋亡、3 - [4,5 - 二甲基噻唑 - 2 - 基] - 2,5 - 二苯基四氮唑溴盐（MTT）和克隆形成试验中，用抗TGFα抗体中和TGFα功能或用MEK1/2抑制剂（PD98059和U0126）抑制MAPK功能，可使照射后的A431和MDA - MB - 231细胞对辐射更加敏感。这些数据表明，破坏TGFα - EGFR - MAPK信号模块是一种减少照射后癌细胞生长和存活的策略。
