Ganglioside Regulation of NFkbeta and Apoptosis

描述：T细胞介导的免疫的产生通常在 癌症患者。我们的研究表明，癌症患者的T细胞 表现出对激活诱导的细胞死亡(AICD)的易感性增加 也表现出NFKB激活的缺陷。这些变化可能会导致 这些患者的T细胞中观察到的免疫功能障碍。神经节苷脂， 已知在某些肿瘤类型中过度表达的基因，可能与 抑制NFKB和提高患者T细胞的敏感度 使细胞发生凋亡。我们和其他人发现，选择神经节苷脂， 包括GD3在内，可以使正常人T细胞对激活诱导的细胞敏感 死亡(AICD)。尽管GD3本身不足以诱导细胞凋亡，但它确实能 抑制抗凋亡基因CLAP-1、CIAP-2和TRAF-2的表达。GD3 也抑制NFkB的激活，这似乎是由于刺激依赖所致 Re1a的降解。NFKB对细胞凋亡的诱导和抑制作用 活动依赖于caspase，因为这两个事件都被caspase阻止 抑制剂Z-VAD-fmk和DEVD-frnk。根据我们的发现，我们假设 癌症患者对肿瘤的免疫力下降是由于 T细胞内促凋亡和抗凋亡蛋白的水平。 包括GD3在内的神经节苷脂通过抑制 抗凋亡基因表达(CIAP/TRAF)。减少的表达 CIAP/TRAF允许caspase活性增加，导致刺激依赖 RELA降解，进一步抑制抗凋亡基因的表达和 诱导细胞凋亡。目标1将定义GD3如何调节抗凋亡基因 在T细胞中表达，并将评估这些分子在 保护细胞免受NFKB抑制和凋亡。初步实验将 确定GD3抑制抗细胞凋亡基因表达的程度 这通常会保护T细胞不受凋亡的影响。实验还将确定 GD3改变稳态抗细胞凋亡水平的机制 基因，包括拍手和TRAF。研究还将测试 抗细胞凋亡基因(cIAPs/traf)的过度表达对Rela的影响 GD3处理的T细胞降解、caspase激活及对AICD的敏感性 细胞。目标2将确定GD3抑制T细胞中NFKB的机制 细胞，以及这种抑制与抗凋亡基因的关系 Expression和AICD。GD3在调节T细胞半胱氨酸氨基转移酶活性中的作用 将通过确定哪些caspase被激活来对细胞进行评估 至GD3，T细胞活化或两者兼而有之。这些激活的半胱氨酸天冬氨酸酶在 然后，通过阻断选定的caspase功能来评估REIA的降解。 在最后一组实验中，REIA将过度表达以确定其 对GD3抑制的抗细胞凋亡基因表达和血管紧张素转换酶的保护作用 使细胞对AICD产生抗性。这些研究将为我们深入了解 神经节苷脂抑制NFKB和生存基因的表达，导致T细胞增加 细胞对细胞凋亡的敏感性。

DESCRIPTION: The generation of T cell mediated immunity is often impaired in cancer patients. Our studies have shown that T cells from cancer patients display increased susceptibility to activation induced cell death (AICD) and also exhibit a defect in NFKB activation. These alterations may contribute to the immune dysfunction observed in T cells from these patients. Gangliosides, which are known to be overexpressed in certain tumor types, may be responsible for the suppression of NFKB and for the increased sensitivity of patient T cells to apoptosis. We and others have found that select gangliosides, including GD3, can sensitize normal human T cells to activation induced cell death (AICD). Although GD3 alone is not sufficient to induce apoptosis, it does inhibit expression of the anti-apoptotic genes clAP-1, cIAP-2 and TRAF-2. GD3 also inhibits NFkB activation, which appears to be due to stimulus-dependent degradation of Re1A . The induction of apoptosis and suppression of NFKB activity are caspase dependent, since both events are blocked by the caspase inhibitors Z-VAD-fmk and DEVD-frnk. Based on our findings, we hypothesize that immunity to tumors is diminished in cancer patients due to an imbalance between the intracellular levels of pro- versus anti-apoptotic proteins in T cells. Gangliosides including GD3 contribute to this imbalance by suppressing anti-apoptotic gene expression (cIAP/TRAF). The reduced expression of cIAPs/TRAFs allows increased caspase activity, leading to stimulus dependent RelA degradation, further depressing anti-apoptotic gene expression and inducing apoptosis. Aim 1 will define how GD3 regulates anti-apoptotic gene expression in T cells, and will assess the role of those molecules in protecting cells from NFKB inhibition and apoptosis. Initial experiments will determine the extent to which GD3 inhibits the anti-apoptotic gene expression that normally protects T cells from apoptosis. Experiments will also determine the mechanism by which GD3 alters the steady state levels of antiapoptotic genes, including clAPs, and TRAFs. Studies will also test the impact that overexpression of the antiapoptotic genes (cIAPs/TRAFs) has on RelA degradation, caspase activation, and sensitivity to AICD in GD3-treated T cells. Aim 2 will determine the mechanism by which GD3 inhibits NFKB in T cells, and the relationship of that inhibition to anti-apoptotic gene expression and to AICD. The role of GD3 in modulating caspase activity in T cells will be assessed by identifying which caspases are activated in response to GD3, T cell activation or both. The role these activated caspases play in degradation of ReIA will then be assessed by blocking select caspase function. In a final set of experiments, ReIA will be overexpressed to determine its protective effect on both GD3-inhibited antiapoptotic gene expression and in rendering cells resistant to AICD. These studies will provide insight into how gangliosides inhibit NFKB and survival gene expression, leading to increased T cell sensitivity to apoptosis.