Regulation of ATPase II and Clearance of Cancer Cells

ATPase II 的调节和癌细胞的清除

• 批准号: 6944110
• 负责人: PROBAL BANERJEE
• 金额: $ 7.86万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6944110
• 关键词: adenosinetriphosphatase; apoptosis; calcium channel; cell line; cell membrane; enzyme activity; enzyme mechanism; gene expression; genetic regulation; genetic transcription; glycosyltransferase; laboratory mouse; lipid transport; microglia; mifepristone; neoplastic cell; phagocytosis; phosphatidylserines; protein protein interaction; small interfering RNA; transfection /expression vector; voltage gated channel

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.

描述（由申请人提供）：在称为吞噬作用的自然过程中，多细胞生物体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损的细胞。这种吞噬细胞，例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特异性地结合至死亡细胞，其中一种是磷脂酰丝氨酸（PS）。PS通常位于质膜的内小叶，在垂死细胞中翻转到外小叶，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，既是Mg 2 +-/-ATP酶，也是氨基磷脂移位酶（APTL），将PS移位到质膜的内小叶。这一过程显然超越了另外两种酶的作用，即乱序酶（scramblase）（将所有磷脂分子双向移位）和翻转酶（floppase）（将磷脂从内小叶非常缓慢地移位到外小叶）。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，导致PS的外化和吞噬作用。一个P型ATP酶，ATP酶II，具有惊人的相似性，APTL和反义ATP酶II的cDNA转染引起PS外化的性质。ATP酶II mRNA的表达显示出组织特异性，在脑和骨骼肌中观察到最高水平的表达。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。在我们当前的项目中，我们将进一步分析ATP酶II基因的启动子，以研究赋予该细胞表达类型特异性的序列元件。此外，在我们早期的研究中，我们观察到ATP酶II的稳定过表达导致海马神经元衍生细胞系HN 2中出现混合的电压门控钙通道，该细胞系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基（1B）的诱导表达。我们将在HN 2细胞中制备和测试ATP酶II的诱导表达系统，以验证这种诱导的钙通道表达是由于反式激活的基因表达还是ATP酶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ATP酶II转染导致PS分子外化，我们将使用这一概念来设计和测试一种新的策略，通过吞噬作用去除癌细胞，而不试图用抗代谢物杀死它们。在该策略中，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的ATP酶II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质的启动子来实现，所述高度癌细胞选择性蛋白质例如（1-6）N乙酰葡糖胺基转移酶V（GnT-V）。去除癌细胞中的ATP酶II将导致PS外化和这些细胞的吞噬去除。研究ATP酶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用途将是我们项目的总体目标。

项目成果

Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter.

无 TATA 的人 ATPase II 启动子的分离、测序和功能分析。

• DOI: 10.1016/j.bbaexp.2005.02.007
• 发表时间: 2005
• 期刊: Biochimica et biophysica acta
• 作者: Sobocki,Tomasz;Jayman,Farah;Sobocka,MalgorzataB;Duchatellier,Ruth;Banerjee,Probal
• 通讯作者: Banerjee,Probal

Clozapine functions through the prefrontal cortex serotonin 1A receptor to heighten neuronal activity via calmodulin kinase II-NMDA receptor interactions.

• DOI: 10.1111/j.1471-4159.2011.07565.x
• 发表时间: 2012-02
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Purkayastha S;Ford J;Kanjilal B;Diallo S;Del Rosario Inigo J;Neuwirth L;El Idrissi A;Ahmed Z;Wieraszko A;Azmitia EC;Banerjee P
• 通讯作者: Banerjee P

Atp8a1 deficiency is associated with phosphatidylserine externalization in hippocampus and delayed hippocampus-dependent learning.

• DOI: 10.1111/j.1471-4159.2011.07543.x
• 发表时间: 2012-01
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Levano K;Punia V;Raghunath M;Debata PR;Curcio GM;Mogha A;Purkayastha S;McCloskey D;Fata J;Banerjee P
• 通讯作者: Banerjee P

Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene.

无 TATA 的鼠 ATPase II 启动子的分离、测序和功能分析以及 ATPase II 基因的结构分析。

• DOI: 10.1016/j.bbaexp.2006.11.007
• 发表时间: 2007
• 期刊: Biochimica et biophysica acta
• 作者: Sobocki,Tomasz;Jayman,Farah;Sobocka,MalgorzataB;Marmur,JonathanD;Banerjee,Probal
• 通讯作者: Banerjee,Probal