Choi W S, Canzoniero L M, Sensi S L, O'Malley K L, Gwag B J, Sohn S, Kim J E, Oh T H, Lee E B, Oh Y J
Department of Biology, Yonsei University College of Science, Seoul, 120-749, Korea.
Exp Neurol. 1999 Sep;159(1):274-82. doi: 10.1006/exnr.1999.7133.
To further characterize MPP(+)-induced cell death and to explore the role of Bcl-2-related proteins in this death paradigm, we utilized a mesencephalon-derived dopaminergic neuronal cell line (MN9D) stably transfected with human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Delta22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP(+) underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP(+)-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 microM) or Boc-aspartyl(OMe)-fluoromethylketone (50-200 microM) did not attenuate MPP(+)-induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP(+)-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP(+) treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP(+) treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP(+)-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP(+) induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP(+)-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells.
为了进一步明确1-甲基-4-苯基吡啶离子（MPP(+)）诱导的细胞死亡特征，并探究Bcl-2相关蛋白在这种死亡模式中的作用，我们利用了一种稳定转染人bcl-2（MN9D/Bcl-2）、其C末端缺失突变体（MN9D/Bcl-2Delta22）、小鼠bax（MN9D/Bax）或对照载体（MN9D/Neo）的中脑源性多巴胺能神经元细胞系（MN9D）。通过电子显微镜和TUNEL分析确定，暴露于MPP(+)的MN9D/Neo细胞经历了一种细胞死亡，其特征为线粒体肿胀和不规则散布的异染色质，且无伴随的DNA片段化。然而，通常在坏死中见到的细胞肿胀并未出现。为了检查与MPP(+)诱导的细胞死亡相关的生化事件，进行了各种分析。添加广谱半胱天冬酶抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（50 - 400 microM）或Boc-天冬氨酸(OMe)-氟甲基酮（50 - 200 microM）并不能减轻MPP(+)诱导的细胞死亡，而相同处理可保护MN9D/Neo细胞免受星形孢菌素诱导的凋亡性细胞死亡。用大分子合成抑制剂如放线菌酮、依米丁或放线菌素D同时处理可阻断MPP(+)诱导的细胞死亡，这表明如在许多凋亡性细胞死亡中所证明的那样，需要新的蛋白质合成。通过荧光探针Fura-2监测，MPP(+)处理后24小时内MN9D/Neo细胞的胞质钙水平未发生变化。蛋白质印迹分析表明，促凋亡蛋白Bax的表达水平在MPP(+)处理后未显著改变。在这种死亡模式中，Bcl-2的过表达而非其C末端缺失突变体可减轻MPP(+)诱导的细胞死亡，而Bax的过表达则无作用。综上所述，这些数据表明：（i）MPP(+)诱导一种既类似于凋亡又类似于坏死的独特细胞死亡形式；（ii）全长Bcl-2通过一种依赖于从头蛋白质合成但独立于MN9D细胞中胞质钙变化、Bax表达和/或半胱天冬酶激活的机制来对抗MPP(+)诱导的形态学变化和细胞死亡。
