Analysis of biological role and molecular mechanisms of autophagic cell death

细胞程序性死亡在后生动物的正常发育和生理过程中是一个至关重要的过程：它可以分为几类，包括I类(细胞凋亡)和II类(自噬死亡)。BCI-2家族蛋白是一类典型的细胞凋亡调节因子，该家族中的多结构域促凋亡分子，如BAX和BAK，起着线粒体通道的作用，在其上汇聚了多种凋亡信号。虽然Bax/Bak双基因敲除(DKO)小鼠的胚胎成纤维细胞对凋亡具有抵抗力，但我们发现这些细胞在死亡刺激后仍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被包括3-甲基腺嘌呤在内的自噬抑制剂抑制，并且依赖于自噬蛋白APG5和Beclin 1。我们将这些研究扩展到分析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被加入JNK抑制剂或表达显性负突变的JNK所抑制。然而，氨基酸耗竭诱导的DKO细胞死亡不依赖于JNK。本研究还获得了ATG5/BAX/BAK(TKO)小鼠胚胎，发现细胞凋亡和自噬细胞死亡是相互补偿的。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.