Hansen Katharina, Wagner Bettina, Hamel Wolfgang, Schweizer Michaela, Haag Friedrich, Westphal Manfred, Lamszus Katrin
Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistrasse, Hamburg, Germany.
J Neurochem. 2007 Oct;103(1):259-75. doi: 10.1111/j.1471-4159.2007.04753.x. Epub 2007 Jul 17.
The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.
神经营养因子受体原肌球蛋白相关激酶A（TrkA）及其配体神经生长因子（NGF）在星形细胞瘤中表达，并且TrkA表达与恶性程度呈负相关。我们推测TrkA表达可能使胶质母细胞瘤细胞具有生长劣势。为了分析TrkA的功能和信号传导，我们将人TrkA cDNA转染到人胶质母细胞瘤细胞系G55中。我们获得了三个稳定克隆，所有克隆对NGF均有明显的细胞质空泡化反应并随后发生细胞死亡。分析细胞死亡机制时，我们排除了凋亡和细胞衰老。相反，我们发现了自噬的几个迹象：电子显微镜显示典型的自噬泡；吖啶橙染色显示酸性囊泡细胞器；用巴弗洛霉素A1阻止酸性囊泡细胞器的酸化；细胞在G2/M期停滞；LC3的加工增加；自噬抑制剂3-甲基腺嘌呤可防止空泡化；未检测到半胱天冬酶激活。我们进一步发现，ERK和c-Jun N端激酶的激活而非p38的激活参与了自噬泡的形成。总之，我们确定自噬是NGF诱导细胞死亡的新机制。我们的研究结果表明，人胶质母细胞瘤中TrkA的激活在治疗上可能是有益的，特别是因为目前使用的几种化疗药物也诱导自噬性细胞死亡。
