Regulation of ATPase II and Clearance of Cancer Cells

描述（由申请人提供）：在称为吞噬作用的自然过程中，多细胞生物在称为吞噬细胞的清道夫细胞的帮助下去除不需要的或受伤的细胞。这些吞噬细胞，如外周系统中的巨噬细胞和大脑中的小胶质细胞，通过某些细胞表面分子特异性地与垂死细胞结合，其中一种是磷脂酰丝氨酸（PS）。PS通常位于质膜的内小叶，在死亡细胞中转移到外小叶，并被位于吞噬细胞上的PS受体识别。在健康细胞中，一种双功能酶，即Mg2+-/- atp酶和氨基磷脂转位酶（APTL），可将PS转运到质膜的内小叶。这一过程显然超越了另外两种酶的作用，即转录酶（双向转运所有磷脂分子）和转录酶（非常缓慢地将磷脂从内叶转运到外叶）。许多早期的研究表明，通过抑制APTL来抵消APTL、scramblase和floppase之间的平衡会导致PS外化和吞噬。一种p型atp酶，ATPase II，与APTL具有惊人的相似性，转染反义ATPase II cDNA可引起PS的外化。ATPase II mRNA的表达具有组织特异性，在大脑和骨骼肌中表达水平最高。我们已经分离出atp酶II启动子，并表明它在荧光素酶报告基因检测中显示出相当大的组织特异性。在我们目前的项目中，我们将进一步分析atp酶II基因的启动子，以研究赋予这种细胞类型特异性表达的序列元件。此外，在我们早期的研究中，我们观察到ATPase II的稳定过表达会导致海马神经元来源细胞系HN2中出现混合的电压门控钙通道，而这些钙通道通常不显示任何钙电流。同时，n型通道的通道形成亚基（1B）的诱导表达也被观察到。我们将在HN2细胞中制备和测试ATPase II的诱导表达系统，以验证这种诱导钙通道的表达是由于反激活的基因表达还是由于ATPase II与钙通道蛋白之间的蛋白-蛋白相互作用。最后，由于反义atp酶II转染导致PS分子外化，我们将利用这一概念设计和测试一种通过吞噬去除癌细胞的新策略，而不试图用抗代谢物杀死它们。在这个策略中，我们将通过使用atp酶II特异性小抑制RNA （siRNA）选择性地消融癌细胞中atp酶II的表达。siRNA在癌细胞中的选择性表达将通过使用高度癌细胞选择性蛋白的启动子来实现，例如（1-6 N乙酰氨基葡萄糖转移酶V (GnT-V)）。癌细胞中atp酶II的缺失会导致PS外化和吞噬细胞的清除。研究ATPase II的表达机制，分析其在钙通道诱导中的作用，并评估其在设计新的癌细胞去除策略中的应用将是我们项目的总体目标。

DESCRIPTION (provided by applicant): In the natural process known as phagocytosis a multicellular organism removes unwanted or injured cells with the help of scavenger cells, termed phagocytes. Such phagocytic cells, e.g. the macrophages in the peripheral system and the microglia in the brain, specifically bind to the dying cells through certain cell-surface molecules, one of which is phosphatidylserine (PS). PS, which normally resides in the inner-leaflet of the plasma membrane, flip flops to the outer leaflet in the dying cells and is recognized by PS receptors located on phagocytic cells. In healthy cells a bifunctional enzyme, which is both a Mg2+-/-ATPase and also an aminophospholipid translocase (APTL), translocates PS to the inner leaflet of the plasma membrane. This process apparently overrides the effect of two other enzymes, the scramblase (which bidirectionally translocates all phospholipids molecules) and the floppase (which very slowly translocates phospholipids from the inner leaflet to the outer). Many earlier studies have shown that offsetting the balance among APTL, scramblase, and floppase by inhibiting APTL results in externalization of PS and phagocytosis. A P-type ATPase, ATPase II, bears striking similarity in properties to APTL and transfection of antisense ATPase II cDNA causes externalization of PS. Expression of ATPase II mRNA shows tissue-specificity, with the highest levels of expression observed in the brain and the skeletal muscle. We have isolated the ATPase II promoter and showed that it displays considerable tissue specificity in luciferase reporter assays. In our current project we will further analyze the promoter of the ATPase II gene to study the sequence elements that confer this cell type-specificity of expression. Also, in our earlier studies, we had observed that stable overexpression of ATPase II causes appearance of mixed, voltage-gated calcium channels in a hippocampal neuron-derived cell line, HN2, which normally does not display any calcium current. In parallel, induced expression of the channel-forming subunit, (1B, of the N-type channels was also observed. We will prepare and test an inducible expression system for ATPase II in the HN2 cells in order to verify if this induced expression of calcium channels was due to transactivated gene expression or a protein-protein interaction between ATPase II and the calcium channel proteins. Finally, since antisense ATPase II transfection causes externalization of PS molecules, we will use this concept to design and test a novel strategy of removing cancer cells through phagocytosis without attempting to kill them with antimetabolites. In this strategy we will selectively ablate ATPase II expression in the cancer cells by using ATPase II-specific small inhibitory RNA (siRNA). Selective expression of the siRNA in cancer cells will be achieved through the use of a promoter for a highly cancer cell-selective protein, e.g. the (1-6 N acetyl glucosaminyl transferase V (GnT-V). Abrogation of ATPase II in the cancer cells would cause PS externalization and phagocytic removal of these cells. Studying the mechanism of ATPase II expression, analyzing its role in the induction of calcium channels, and evaluating its use in designing a new strategy for the removal of cancer cells will be the overall goal of our project.