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学中的一个关键过程：它可以分为几类，包括I型（凋亡）和II型（自噬死亡）。Bcl-2蛋白家族是细胞凋亡的良好表征的调节剂，并且该家族的多结构域促细胞凋亡成员，如Bax和巴克，作为多种细胞凋亡信号会聚的线粒体网关起作用。尽管Bax/巴克双基因敲除（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然以具有自噬特征的非凋亡方式死亡。此外，我们还发现DKO细胞的非凋亡性死亡可被自噬抑制剂（包括3-甲基腺嘌呤）抑制，并依赖于自噬蛋白APG 5和Beclin 1。我们将这些研究扩展到自噬性死亡的分子机制，发现JNK的激活在自噬性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细胞自噬性死亡过程中观察到JNK活化，（2）这种形式的细胞死亡通过加入JNK抑制剂或表达JNK的显性阴性突变体而受到抑制。然而，氨基酸消耗诱导的DKO细胞死亡不依赖于JNK。本研究还制备了ATG 5/Bax/巴克（TKO）小鼠胚胎，发现凋亡与自噬性细胞死亡是相互补偿的。此外，我们还发现一些癌细胞在自噬相关基因上发生了突变。

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.