Apoptosis driven by elastase inhibitor: new approach to detection and study.

描述(申请人提供)：已发现多种导致癌细胞凋亡的途径。为了了解在每种特定情况下控制细胞消除的机制，有必要有能够区分不同凋亡途径的探针。最新发现的一种细胞凋亡机制是弹性酶抑制因子诱导的细胞凋亡。然而，这一特殊的通路仍然缺乏组织切片格式的具体可视化工具，而组织切片格式与病理学研究最相关。单核细胞中性粒细胞弹性蛋白酶抑制物(MNEI)是该途径的主要成员，负责其激活。它的作用是自我转化为一种活性核酸酶，并能迅速处理肿瘤细胞。目前，促进脑瘤这种独特转变的条件还不完全清楚。在很大程度上，这是因为在组织切片和活细胞培养中，缺乏选择性标记MNEI驱动的细胞凋亡的特定工具。在缺乏针对这一凋亡途径的特定原位方法的情况下，目前对它的研究主要是使用大量的生化方法，这些方法在不同的病理标本中的应用价值有限。在这个项目中，我们将克服这一障碍，开发新的使能成像技术。我们将应用新的探针来可视化MNEI诱导的胶质母细胞瘤(GBM)中的细胞凋亡。具体目的：1.建立首个选择性检测弹性酶抑制物驱动的组织切片中细胞凋亡途径的方法学。该方法将标记这一途径的关键事件，并显示单核细胞中性粒细胞弹性酶抑制物(MNEI)转化为活性DNA酶后特有的DNA切割活性。在胶质母细胞瘤切片中测试这种新开发的成像工具。2.建立活体细胞培养中弹性酶抑制物介导的细胞凋亡途径的选择性标记新方法。新的针对MNEI途径的荧光传感器只有在检测到这一凋亡途径的特定标记物后才会产生荧光。该项目将为一般的细胞凋亡研究，特别是癌症研究引入使能技术。它们在胶质母细胞瘤中的应用将为这些肿瘤的临床和研究提供有用的信息，并为开发有效的治疗干预措施提供有用的信息。

DESCRIPTION (provided by applicant): Multiple pathways leading to apoptosis of cancer cells have been discovered. To understand the mechanisms controlling cell elimination in each specific situation, it is necessary to have probes which can distinguish between different apoptotic routes. One of the most recently identified apoptotic programs is the elastase inhibitor-driven apoptosis. However, this particular pathway still lacks specific tools for its visualizationin the tissue section format, which is most relevant for pathology studies. Monocyte neutrophil elastase inhibitor (MNEI) is the main member of this pathway responsible for its activation. It acts by self-converting into an active nuclease and can rapidly dispose of tumor cells. The conditions promoting such a unique transition in brain tumors are not fully understood at this time. To a large extent this is because of the absence of specific tools for selective labeling of MNEI-driven apoptosis in tissue sections and in live cell cultures. In the absence of specific in situ methods for this apoptotic pathway, it is currently studied by using the bulk biochemical approaches which have limited value in heterogeneous pathology samples. In this project we will overcome this obstacle and will develop new enabling imaging technologies. We will apply the new probes to visualize MNEI-driven apoptosis in glioblastoma (GBM). Specific aims: 1. To develop the first methodology for selective detection of the elastase inhibitor- driven apoptotic pathway in tissue sections. The approach will label the key event of this pathway and visualize the characteristic DNA cleavage activity of monocyte neutrophil elastase inhibitor (MNEI) after its transformation into an active DNase. To test this newly developed imaging tool in glioblastoma sections. 2. To develop the new methodology for selective labeling of the elastase inhibitor-driven apoptotic pathway in live cell cultures. The new fluorescent sensors specific for MNEI pathway will produce fluorescence only after they detect a specific marker of this apoptotic route. The project will introduce enabling technologies for apoptosis research in general, and for cancer studies in particular. Their application to glioblastoma will provide information useful for future clinical and research investigations of these tumors, and for the development of effective therapeutic interventions.