GAP JUNCTION INTERCELLULAR COMMUNICATION AND POLYAMINES

描述（由申请人提供）：胶质瘤是胶质细胞来源的肿瘤，多形性胶质母细胞瘤是脑癌中最具侵袭性的形式之一。与正常胶质细胞不同，胶质瘤细胞表达低水平的功能性连接蛋白（Cx）半通道/间隙连接蛋白和高水平的多胺（PA）。抗癌策略中的很多注意力集中在（i）PA或（ii）Cx上，尽管对PA和Cx半通道/间隙连接如何共同相互作用知之甚少。发现胶质瘤细胞中Cx的表达主要在细胞质中，具有非常少的细胞表面表达。尽管如此， 神经胶质瘤细胞之间仍然高度功能性的间隙连接细胞间通讯。目前尚不清楚低水平的细胞表面Cx表达如何允许胶质瘤细胞之间的这种高间隙连接细胞间通讯。我们的初步数据表明：（1）PA消除了Cx43间隙连接/半通道的阳离子阻断，（2）增加了它们的开放概率。基于这些发现，我们推测，高水平的PA存在于胶质瘤细胞增强Cx43通道开放和增强间隙连接细胞间通讯。因此，通过Cx43间隙连接/半通道的细胞间信号的传播将被提高。另一方面，最近显示，细胞内PA阻断Cx40间隙连接，表明PA对不同类型的Cx具有不同的作用。目前，还没有关于PA和Cxs（Cx43除外）在胶质细胞中的数据。本研究将探讨正常和恶性胶质细胞中多胺和Cx半通道/缝隙连接之间的相互作用。为了验证我们的假设，我们提出了以下具体目标：具体目标1：确定连接蛋白半通道电流和星形胶质细胞，Cxs转染细胞和Cx43条件敲除小鼠的间隙连接通讯的多胺依赖性。具体目标二：确定胶质瘤细胞中过表达的不同类型的Cxs如何参与间隙连接细胞间通讯，并可能在癌症治疗中发挥作用。意义：这些研究的结果将提供抗癌治疗的两个靶点之间的联系;连接蛋白和多胺。我们将定义多胺对不同Cx的影响。这些知识将帮助我们开发一种有效的策略，在PA水平升高时治疗癌细胞。通过调节Cxs，我们可以调节胶质瘤细胞之间的间隙连接细胞间通讯，以及胶质瘤细胞与肿瘤微环境中正常星形胶质细胞之间的潜在通讯。

DESCRIPTION (provided by applicant): Gliomas are tumors of glial cell origin, with glioblastoma multiforme being one of the most aggressive forms of brain cancer. Unlike normal glia, glioma cells express low levels of functional connexin (Cx) hemichannel/gap junction protein and high levels of polyamines (PA). A lot of attention in the anti-cancer strategies focuses on either (i) PA or (ii) Cxs, although there is not very much known about how PA and Cx hemichannels/gap- junctions co-interact. Expression of Cx in glioma cells is found predominantly in the cytoplasm with very little cell surface expression. In spite of this, there is still highly functional gap junctional intercellular communication between glioma cells. It is unclear how low levels of cell surface Cx expression allow such high gap junctional intercellular communication between glioma cells. Our preliminary data indicate that (1) PA eliminate cationic block of Cx43 gap junctions/hemichannels and (2) increase their open probability. Based on these findings, we hypothesize that high levels of PA present in glioma cells potentiate Cx43 channel opening and enhance gap junctional intercellular communication. As a result, propagation of the intercellular signals through Cx43 gap junctions/hemichannels will be elevated. On the other hand, it was recently shown that intracellular PA block Cx40 gap junctions demonstrating a different effect of PA on different types of Cxs. Currently, there is no data about PA and Cxs (other than Cx43) in glial cells. The present study will examine the interaction between polyamines and Cx hemichannels/gap junctions in normal and malignant glia. To test our hypothesis we propose the following specific aims: Specific Aim 1: To determine the polyamine dependence of connexin hemichannel currents and gap junction communication in astrocytes, Cxs transfected cells and Cx43 conditional knock-out mice. Specific Aim 2: To determine how different types of Cxs overexpressed in glioma cells participate in gap junction intercellular communication and may contribute during cancer treatment. Significance: The results of these studies will provide a link between two targets of anti-cancer therapy; connexins and polyamines. We will define the effects polyamines on the different Cxs. This knowledge will help us to develop an effective strategy to treat cancer cells when the levels of PA are elevated. By regulation of Cxs, we can modulate gap junctional intercellular communication between glioma cells and potentially communication between glioma cells and the normal astrocytes in the tumor microenvironment.