Metabolic Mechanisms for Programmed Cell Death

程序性细胞死亡的代谢机制

• 批准号: 7338799
• 负责人: JAMES M PHANG
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7338799
• 关键词: Metabolic; Mechanisms; Programmed; Cell; Death

In previously published studies, we have shown that overexpression of POX activates the intrinsic (mitochondrial) pathway for apoptosis. Features of this effect include DNA fragmentation, increased sub-Go/G1 fraction on flow cytometry, release of cytochrome c into cytosol, and activation of caspase 9. The mechanism of this effect is due to the generation of proline-dependent superoxide which was assayed using hydroethidine. Importantly, the co-expression of, MnSOD which is localized in mitochondria, abolished the POX-dependent apoptotic effects. We asked whether the extrinsic (death receptor) pathway is also affected by overexpression of POX. We found that POX expression upregulated the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), DR5 and activated caspase-8, a caspase specifically activated through the death receptor pathway. Importantly, apoptosis measured by flow cytometry was partially inhibited by Z-IETD-FMK, a specific inhibitor of caspase-8. Furthermore, the mechanism of this effect may be modified by nuclear factor of activated T-cells (NFAT. Additionally, POX expression markedly decreased the phosphorylation of MEK and ERK and this decrease was partially reversed by expression of MnSOD. These data showed that POX may induce apoptosis through both intrinsic and extrinsic pathways and may modulate apoptosis signals induced by p53 or other anti-cancer agents. To test whether this POX-dependent apoptotic mechanism can be translated to animal models, we performed studies using DLD-tet-off-POX xenograft tumors in athymic mice differentially administered Doxycycline (100 micrograms/ml) in their drinking water. First, we injected DLD-tet-off-vector cells (106) into the lumbar region of athymic mice and found that tumors rapidly grew unaffected by Doxycycline. By the end of week 3, all the animals were euthanized because of the size of the tumors. When DLD-tet-off-POX cells were injected, tumor formation was directly related to intake of Doxycycline. In animals on Doxycycline (POX suppressed), all animals (n=22) rapidly formed tumors in a fashion not different from those injected with DLD-tet-off-vector cells and required euthanization by the end week 3. In contrast, animals off Doxycycline (POX induced), had marked retardation of tumor growth. In fact, by the end of week 3, tumors were palpable in only 14% of the animals (3/22). Even by the end of week 5, palpable tumors were found in only 32% (7/22). These studies suggested that POX expression and the resultant proline-dependent apoptosis markedly decreased the formation of tumors in athymic mice. To extend our findings into POX-dependent suppression of specific tumors, we will develop POX transgenic mice and cross them with mice with known tumor susceptibilities to determine whether POX expression affects specific tumor phenotypes.

在以前发表的研究中，我们已经表明，POX的过度表达激活了细胞凋亡的内在（线粒体）途径。这种效应的特征包括DNA片段化、流式细胞术上亚G 0/G1级分增加、细胞色素c释放到胞质溶胶中以及半胱天冬酶9的活化。这种作用的机制是由于脯氨酸依赖性超氧化物的产生，这是使用氢乙啶测定。重要的是，共同表达，MnSOD，这是定位在线粒体，取消POX依赖性凋亡的影响。我们询问是否外在（死亡受体）途径也受到POX过表达的影响。我们发现，POX表达上调肿瘤坏死因子相关凋亡诱导配体（TRAIL），DR 5和激活的caspase-8，caspase特异性激活通过死亡受体途径的表达。重要的是，通过流式细胞术测量的细胞凋亡被半胱天冬酶-8的特异性抑制剂Z-IETD-FMK部分抑制。此外，这种作用的机制可能被活化T细胞核因子（NFAT）修饰。此外，POX的表达显着降低MEK和ERK的磷酸化，这种降低被MnSOD的表达部分逆转。这些数据表明，POX可能通过内源性和外源性途径诱导细胞凋亡，并可能调节p53或其他抗癌药物诱导的细胞凋亡信号。为了测试这种POX依赖性细胞凋亡机制是否可以转化为动物模型，我们在无胸腺小鼠中使用DLD-tet-off-POX异种移植肿瘤进行了研究，这些小鼠在饮用水中差异施用多西环素（100微克/毫升）。首先，我们将DLD-tet-off-vector细胞（106）注射到无胸腺小鼠的腰部，发现肿瘤不受强力霉素的影响而迅速生长。到第3周结束时，由于肿瘤的大小，所有动物都被安乐死。当注射DLD-tet-off-POX细胞时，肿瘤形成与多西环素的摄入直接相关。在使用多西环素（POX抑制）的动物中，所有动物（n=22）以与注射DLD-tet-off-载体细胞的那些动物没有不同的方式快速形成肿瘤，并且在第3周结束时需要安乐死。相比之下，不使用多西环素（POX诱导的）的动物具有肿瘤生长的显著阻滞。事实上，到第3周结束时，仅14%的动物（3/22）可触及肿瘤。即使在第5周结束时，也仅在32%（7/22）中发现可触及的肿瘤。这些研究表明，POX的表达和由此产生的脯氨酸依赖性凋亡显着减少了无胸腺小鼠肿瘤的形成。为了将我们的发现扩展到POX依赖性抑制特定肿瘤，我们将开发POX转基因小鼠，并将其与已知肿瘤易感性的小鼠杂交，以确定POX表达是否影响特定的肿瘤表型。