Pittman R N, Wang S, DiBenedetto A J, Mills J C
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Neurosci. 1993 Sep;13(9):3669-80. doi: 10.1523/JNEUROSCI.13-09-03669.1993.
A model system has been established in which PC12 cells are converted to neuronal-like cells that undergo transcription-dependent cell death following removal of NGF. Nineteen sublines of PC12 cells were tested to establish parameters for making cells dependent on NGF for survival. In most sublines, a relatively small percentage of cells become dependent on NGF for survival, and following removal of NGF, most of the cells begin proliferating in serum-containing medium. In several sublines, however, a significant percentage of cells die following removal of NGF. One of these sublines, PC6-3, can be grown under conditions in which 90% of the cells undergo transcription-dependent cell death following removal of NGF in either serum-free or serum-containing medium. Fourteen hours after removing NGF, 50% of the cells are committed to die, while initial morphological signs of cell death as determined by time-lapse videomicroscopy occur 2-6 hr later and include loss of neurites followed by a 1-3 hr period of active membrane "blebbing" and protrusions. Cell death can be blocked by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, KCl, basic fibroblast growth factor, or dibutryl-cAMP, but not by epidermal growth factor, leupeptin, or the endonuclease inhibitor aurintricarboxylic acid (ATA). Removal of NGF activates an endonuclease that causes nucleosomal laddering of the DNA; however, endonuclease activity does not appear to be required for cell death. In agreement with previous studies (Batistatou and Greene, 1991; Rukenstein et al., 1991) demonstrating that naive PC12 cells undergo transcription-independent cell death when shifted into serum-free medium in the absence of growth factors, all cell lines tested except for one die when cultured in RPMI medium lacking growth factors. DNA fragmentation is a prominent feature of transcription-independent cell death, and death can be blocked with NGF, ATA, and dibutryl-cAMP but not with actinomycin D or KCl. The PC12 model system described here should be useful for identifying cell death genes and for characterizing cellular and molecular events in programmed neuronal cell death.
已建立一种模型系统，其中PC12细胞可转化为神经元样细胞，在去除神经生长因子（NGF）后会发生转录依赖性细胞死亡。对19个PC12细胞亚系进行了测试，以确定使细胞依赖NGF存活的参数。在大多数亚系中，相对较小比例的细胞依赖NGF存活，去除NGF后，大多数细胞开始在含血清培养基中增殖。然而，在几个亚系中，去除NGF后有相当比例的细胞死亡。其中一个亚系PC6-3，可在这样的条件下生长：去除无血清或含血清培养基中的NGF后，90%的细胞会发生转录依赖性细胞死亡。去除NGF 14小时后，50%的细胞注定死亡，而通过延时视频显微镜确定的细胞死亡的初始形态学迹象在2 - 6小时后出现，包括神经突消失，随后是1 - 3小时的活跃膜“起泡”和突起期。细胞死亡可被RNA合成抑制剂放线菌素D、蛋白质合成抑制剂环己酰亚胺、KCl、碱性成纤维细胞生长因子或二丁酰环磷腺苷（dibutryl-cAMP）阻断，但不能被表皮生长因子、亮抑蛋白酶肽或核酸内切酶抑制剂金精三羧酸（ATA）阻断。去除NGF会激活一种核酸内切酶，导致DNA出现核小体梯状条带；然而，核酸内切酶活性似乎并非细胞死亡所必需。与先前的研究（Batistatou和Greene，1991；Rukenstein等人，1991）一致，这些研究表明，在缺乏生长因子的情况下，将未处理的PC12细胞转移到无血清培养基中会发生转录非依赖性细胞死亡，除一个细胞系外，所有测试的细胞系在缺乏生长因子的RPMI培养基中培养时都会死亡。DNA片段化是转录非依赖性细胞死亡的一个突出特征，死亡可被NGF、ATA和二丁酰环磷腺苷阻断，但不能被放线菌素D或KCl阻断。本文所述的PC12模型系统应有助于鉴定细胞死亡基因，并表征程序性神经元细胞死亡中的细胞和分子事件。
