Analysis of biological role and molecular mechanisms of autophagic cell death

程序性细胞死亡是后生动物正常发育和生理的重要过程，可分为I型（细胞凋亡）和II型（自噬性死亡）。BcI-2蛋白家族是细胞凋亡的调控因子，该家族的多结构域促凋亡成员，如Bax和Bak，作为线粒体通道，多种凋亡信号在其上汇聚。虽然Bax/Bak双敲除（DKO）小鼠的胚胎成纤维细胞对凋亡具有抗性，但我们发现这些细胞在死亡刺激后仍以非凋亡方式死亡，具有自噬特征。此外，我们还发现DKO细胞的非凋亡性死亡受到自噬抑制剂（包括3-甲基腺嘌呤）的抑制，并且依赖于自噬蛋白APG5和Beclin 1。我们扩展了这些研究，分析了自噬死亡的分子机制，发现JNK的激活在自噬细胞死亡中起着至关重要的作用。这一结论得到以下事实的支持：(1)在依托泊苷诱导的DKO细胞自噬死亡过程中观察到JNK的激活，(2)这种形式的细胞死亡可以通过添加JNK抑制剂或表达JNK的显性阴性突变体来抑制。然而，氨基酸消耗诱导的DKO细胞死亡不依赖于JNK。这些结果表明，JNK的激活对DKO细胞的自噬死亡至关重要。我们还制作了ATG5/Bax/Bak （TKO）小鼠胚胎，发现细胞凋亡和细胞自噬死亡相互补偿。此外，我们还发现一些癌细胞的自噬相关基因发生了突变。

Programmed cell death is a crucial process in the normal development and physiology of metazoans : it can be divided in several categories including type I (apoptosis) and type II (autophagic death). The BcI-2 family of proteins are well-characterized regulators of apoptosis, and multidomain pro-apoptotic members of this family, such as Bax and Bak, function as a mitochondrial gateway on which a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knock-out (DKO) mice are resistant to apoptosis, we have found that these cells still died in a non-apoptotic fashion with autophagic features after death stimulation. Furthermore, we have also shown that the non-apoptotic death of DKO cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, and was dependent on an autophagy protein APG5 as well as Beclin 1.We have extended these studies to analyze molecular mechanism of the autophagic death, and found that activation of JNK played a crucial role in autophagic cell death. This conclusion was supported by the facts that (1) JNK activation was observed during etoposide-induced autophagic death of DKO cells, (2) this form, of cell death was suppressed by addition of JNK inhibitors or expression of a dominant-negative mutant of JNK. However, amino acid depletion-induced death of DKO cells was not dependent on JNK. These results indicated that JNK activation is crucial for the autophagic death of DKO cells.We also produced ATG5/Bax/Bak (TKO) mice embryo, and found that apoptosis and autophagic cell death compensated each other. Furthermore, we also discovered that some of the cancer cells had mutations in the autophagy-related gene.