Regulation of ATPase II and Clearance of Cancer Cells

描述(申请人提供)：在被称为吞噬作用的自然过程中，多细胞有机体在清道夫细胞(称为吞噬细胞)的帮助下去除不需要的或受损的细胞。这种吞噬细胞，如外周系统中的巨噬细胞和脑中的小胶质细胞，通过某些细胞表面分子与濒死的细胞特异性结合，其中之一是磷脂酰丝氨酸(PS)。PS通常位于质膜的内叶，在垂死的细胞中转到外叶，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，既是镁/三磷酸腺苷酶，也是氨基磷脂转位酶(APTL)，将PS转移到质膜的内叶。这个过程显然超过了另外两种酶的作用，扰乱酶(双向转移所有磷脂分子)和软脂酶(非常缓慢地将磷脂从小叶内部转移到外部)。许多早期的研究表明，通过抑制APTL来抵消APTL、扰乱酶和Floppase之间的平衡会导致PS的外化和吞噬作用。一种P型ATPase，ATPase II，在性质上与APTL有惊人的相似之处，并且反义ATPase II基因的转染导致PS的外化。ATPase II mRNA的表达具有组织特异性，在大脑和骨骼肌中的表达水平最高。我们已经分离了ATPase II启动子，并在荧光素酶报告分析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ATPase 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，我们已经观察到ATPase II的稳定过表达导致在海马神经元衍生细胞系HN2中出现混合的电压门控钙通道，该细胞通常不显示任何钙电流。同时，还观察到N型通道的通道形成亚单位(1B)的诱导表达。我们将在HN2细胞中制备和测试ATPase II的诱导表达系统，以验证这种钙通道的诱导表达是由于反式激活的基因表达，还是由于ATPase II和钙通道蛋白之间的蛋白质-蛋白质相互作用。最后，由于反义ATPase II的转染导致PS分子的外化，我们将利用这一概念来设计和测试一种新的策略，即通过吞噬来移除癌细胞，而不试图用抗代谢药物杀死它们。在这一策略中，我们将通过使用ATPase II特异的小抑制RNA(SiRNA)选择性地阻断癌细胞中ATPase II的表达。在癌细胞中选择性表达siRNA将通过使用高度肿瘤细胞选择性蛋白的启动子，例如(1-6N乙酰氨基葡萄糖转移酶V(GnT-V))。癌细胞中ATPase II的缺失会导致PS外化和吞噬细胞的去除。研究ATPase II的表达机制，分析其在钙通道诱导中的作用，并评估其在设计去除癌细胞的新策略中的作用，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.