Metabolic Mechanisms for Programmed Cell Death

在先前发表的研究中，我们已经表明，POX的过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断裂，流式细胞术中亚go /G1分数增加，细胞色素c释放到细胞质中，以及caspase 9的激活。这种效应的机制是由于产生脯氨酸依赖的超氧化物，用氢乙胺测定。重要的是，位于线粒体中的MnSOD的共表达消除了pox依赖性的凋亡效应。我们询问外源性（死亡受体）通路是否也受POX过表达的影响。我们发现，痘表达上调肿瘤坏死因子相关凋亡诱导配体（TRAIL）、DR5的表达，并激活caspase-8（一种通过死亡受体途径特异性激活的caspase）。重要的是，通过流式细胞术检测到的细胞凋亡被caspase-8特异性抑制剂Z-IETD-FMK部分抑制。此外，这种作用的机制可能与活化t细胞核因子（NFAT）有关。此外，痘表达显著降低了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，痘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。为了测试这种依赖于痘的凋亡机制是否可以转化为动物模型，我们在胸腺小鼠的饮水中不同地给予多西环素（100微克/毫升），使用dld -off- pox异种移植肿瘤进行了研究。首先，我们将dld - et-脱载体细胞（106）注射到胸腺小鼠的腰椎区域，发现肿瘤迅速生长，不受强力霉素的影响。到第三周结束时，由于肿瘤的大小，所有的动物都被安乐死了。当注射dld - et-off- pox细胞时，肿瘤的形成与多西环素的摄入直接相关。在注射强力霉素（痘抑制）的动物中，所有动物（n=22）迅速形成肿瘤的方式与注射dld -t -脱载体细胞的动物没有什么不同，并需要在第3周结束时实施安乐死。相比之下，停用强力霉素（痘诱导）的动物有明显的肿瘤生长迟缓。事实上，在第3周结束时，只有14%的动物（3/22）可触及肿瘤。即使在第5周结束时，可触及的肿瘤也只有32%（7/22）。这些研究表明，在胸腺小鼠中，痘的表达和由此产生的脯氨酸依赖性细胞凋亡显著减少了肿瘤的形成。为了将我们的发现扩展到特异性肿瘤的POX依赖抑制，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表达是否影响特异性肿瘤表型。

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.