Buron Nelly, Micheau Olivier, Cathelin Séverine, Lafontaine Pierre-Olivier, Creuzot-Garcher Catherine, Solary Eric
INSERM (Institut National de la Santé et de la Recherche Médicale) U517, Faculty of Medicine, Dijon, France.
Invest Ophthalmol Vis Sci. 2006 Oct;47(10):4221-30. doi: 10.1167/iovs.05-1460.
To determine the molecular mechanisms of conjunctival cell death on exposure to the quaternary ammonium preservative benzalkonium chloride (BAC) and ultraviolet (UV) irradiation.
Chang conjunctival cells, either wild-type or stably transfected with various constructs encoding antiapoptotic molecules or transiently transfected with siRNA targeting the beclin-1 gene, were exposed to BAC or UV radiation Cell death was analyzed morphologically with fluorescence and electron microscopy, and molecular mechanisms of death were studied by using immunofluorescence, cell fractionation, caspase substrates, and immunoblot analysis, with or without immunoprecipitation. The main results were controlled in IOBA-NHC cells.
Both agents induced cytochrome c release from the mitochondria, caspase activation, and nuclear chromatin condensation, suggesting caspase-dependent apoptosis. These events are prevented by stable expression of Bcl-2 protein. Both agents also induced a redistribution of Fas in plasma membrane rafts and the Fas-ligand-independent formation of a death-inducing complex leading to caspase-8 activation. Stable expression of either a dominant negative construct of Fas-associated death domain (FADD) or the long or short isoform of FADD-like interleukin-1-beta-converting enzyme inhibitory protein (FLIP) inhibited caspase-8 activation in response to both UV radiation and BAC. However, these proteins, as well as permeant peptides and baculovirus p35 caspase-inhibitors, delayed more efficiently the UV irradiation-induced than the BAC-induced nuclear chromatin condensation. BAC specifically activated a caspase-independent pathway by inducing the mitochondrial release of apoptosis-inducing factor. BAC-treated cells contain autophagosomes/autolysosomes, a characteristic feature of autophagy, and siRNA-mediated downregulation of the beclin-1 gene, whose product is crucial for autophagy, increases BAC toxicity.
UV irradiation induces typical, caspase-dependent cell death, whereas death induced by BAC associates features of caspase-dependent and -independent apoptosis counteracted by an autophagic process.
确定结膜细胞在暴露于季铵防腐剂苯扎氯铵（BAC）和紫外线（UV）照射时发生细胞死亡的分子机制。
将野生型或稳定转染了各种编码抗凋亡分子构建体的Chang结膜细胞，或瞬时转染了靶向beclin-1基因的小干扰RNA（siRNA）的细胞，暴露于BAC或UV辐射下。通过荧光和电子显微镜对细胞死亡进行形态学分析，并使用免疫荧光、细胞分级分离、半胱天冬酶底物以及免疫印迹分析（有或没有免疫沉淀）来研究死亡的分子机制。主要结果在IOBA-NHC细胞中得到验证。
两种试剂均诱导线粒体释放细胞色素c、半胱天冬酶激活以及核染色质浓缩，提示半胱天冬酶依赖性凋亡。这些事件可通过Bcl-2蛋白的稳定表达来预防。两种试剂还诱导Fas在质膜脂筏中重新分布，并导致Fas配体非依赖性死亡诱导复合物的形成，从而导致半胱天冬酶-8激活。Fas相关死亡结构域（FADD）的显性负性构建体或FADD样白细胞介素-1-β转换酶抑制蛋白（FLIP）的长或短异构体的稳定表达，均可抑制UV辐射和BAC诱导的半胱天冬酶-8激活。然而，这些蛋白以及渗透性肽和杆状病毒p35半胱天冬酶抑制剂，对UV照射诱导的核染色质浓缩的延迟作用比BAC诱导的更有效。BAC通过诱导线粒体释放凋亡诱导因子，特异性激活了一条不依赖半胱天冬酶的途径。经BAC处理的细胞含有自噬体／自溶酶体，这是自噬的一个特征，而siRNA介导的beclin-1基因下调（其产物对自噬至关重要）会增加BAC的毒性。
UV照射诱导典型的、半胱天冬酶依赖性细胞死亡，而BAC诱导的死亡则具有半胱天冬酶依赖性和非依赖性凋亡的特征，并被自噬过程所抵消。
