Regulation of TRAIL-induced apoptosis

描述（申请人提供）：TRAIL （tumor necrosis factor-related apoptosis inducing ligand，肿瘤坏死因子相关凋亡诱导配体）是一种细胞因子，在许多癌细胞中触发凋亡细胞死亡，但在正常的未转化细胞中不触发。由于这种对癌细胞的选择性毒性，TRAIL是一种有吸引力的抗癌治疗剂。不同癌细胞和正常细胞对TRAIL的敏感性部分受结合TRAIL但不能发出凋亡信号的抑制受体的表达调节。然而，在癌细胞和正常细胞中，抑制/受体控制trail诱导的细胞死亡的机制知之甚少。本项目的长期目标是阐明TRAIL抑制受体控制癌细胞和正常细胞死亡的分子机制。在我们的初步研究中，我们发现死亡受体TRAIL-R2和抑制受体TRAIL-R4以一种不依赖于配体的方式相互作用，这种相互作用是TRAIL-R4抑制细胞凋亡的核心。基于这些结果，我们建议研究不同癌细胞中TRAIL-R2和TRAIL-R4之间的预配体相互作用是否与它们对trail诱导的细胞凋亡的抗性相关。该提案的第二个目标是研究“脂筏”在调节预配体TRAIL-R2/TRAIL-R4组装中的作用。最后，我们将研究预组装的TRAIL-R2/TRAIL-R4复合物是否通过激活促存活转录因子NF κ b来抑制细胞凋亡。为了实现这些目标，我们将通过生化共免疫沉淀、化学交联和流式细胞术荧光共振能量转移（FRET）检测TRAIL-R2和TRAIL-R4在癌细胞和正常细胞中的相互作用。此外，我们将研究这些相互作用在不同的膜微域。此外，我们将通过RNA干扰和转染检测TRAIL-R4在不同癌细胞和正常细胞中的表达，以评估TRAIL-R4在调节NF-kappa B活化中的作用。摘要：TRAIL是一种很有前途的抗癌药物，它可以选择性地杀死癌细胞，而不是正常细胞。此外，TRAIL可与放疗或化疗协同，增强对癌细胞的杀伤。我们的实验也可能提供一个有用的诊断工具来确定癌症对TRAIL或激动剂TRAIL抗体治疗的反应。

DESCRIPTION (provided by applicant): TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a cytokine that triggers apoptotic cell death in many cancer cells, but not normal untransformed cells. Because of this selective toxicity to cancer cells, TRAIL is an attractive anti-cancer therapeutic agent. The sensitivity of different cancer cells and normal cells to TRAIL is partly regulated by expression of inhibitory receptors that bind TRAIL but cannot signal for apoptosis. However, little is known about the mechanism by which the inhibition/ receptors controls TRAIL-induced cell death in cancer cells and normal cells. The long term goal of this project is to elucidate the molecular mechanism by which the TRAIL inhibitory receptors control cell death in cancer cells and normal cells. In our preliminary studies, we found that the death receptor TRAIL-R2 and the inhibitory receptor TRAIL-R4 interact with each other in a ligand-independent manner and that this interaction is central to the inhibition of apoptosis by TRAIL-R4. Based on these results, we propose to examine whether pre-ligand interaction between TRAIL-R2 and TRAIL-R4 in different cancer cells correlates with their resistance to TRAIL-induced apoptosis. A second goal of the proposal is to examine the role of "lipid rafts" in regulating pre-ligand TRAIL-R2/TRAIL-R4 assembly. Finally, we will examine whether pre-assembled TRAIL-R2/TRAIL-R4 complex inhibits apoptosis by activating the pro-survival transcription factor NF kappa B. To achieve these goals, we will examine TRAIL-R2 and TRAIL-R4 interaction in cancer cells and normal cells by biochemical co-immunoprecipitations, chemical crosslinking and flow cytometric fluorescence resonance energy transfer (FRET) assays. In addition, we will examine these interactions in different membrane microdomains. Furthermore, we will examine modulate TRAIL-R4 expression in different cancer cells and normal cells by RNA interference and transfection to evaluate the role of TRAIL-R4 in regulating NF-kappa B activation. Lay summary: TRAIL is a promising anti-cancer agent that selectively kills cancer cells, but not normal cells. Moreover, TRAIL can synergize with radiotherapy or chemotherapy to enhance killing of cancer cells. Our experiments may also provide a useful diagnostic tool to determine response of cancers to treatments involving TRAIL or agonist TRAIL antibodies.