GAP JUNCTION INTERCELLULAR COMMUNICATION AND POLYAMINES

描述(申请人提供)：胶质瘤是胶质细胞起源的肿瘤，多形性胶质母细胞瘤是最具侵袭性的脑癌之一。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/缝隙连接蛋白和高水平的多胺(PA)。许多抗癌策略的注意力集中在(I)PA或(Ii)CXS上，尽管关于PA和CX半通道/缝隙连接是如何相互作用的还不是很清楚。CX在胶质瘤细胞中的表达主要在细胞质中，细胞表面的表达很少。尽管如此，还是有 胶质瘤细胞间的缝隙连接通讯仍然具有很高的功能。目前尚不清楚细胞表面Cx的低水平表达如何允许胶质瘤细胞之间如此高的缝隙连接细胞间通讯。我们的初步数据表明：(1)PA消除了Cx43缝隙连接/半通道的阳离子阻塞，(2)增加了它们的开放几率。基于这些发现，我们假设胶质瘤细胞中存在高水平的PA增强Cx43通道的开放，并增强缝隙连接细胞间的通讯。因此，细胞间信号通过Cx43缝隙连接/半通道的传播将被提升。另一方面，最近的研究表明，细胞内PA阻断Cx40缝隙连接，表明PA对不同类型的CXS的作用不同。目前，除了Cx43外，还没有关于PA和CXS在神经胶质细胞中的相关数据。本研究将在正常和恶性胶质细胞中检测多胺和Cx半突触/缝隙连接的相互作用。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：确定星形胶质细胞、CXS转基因细胞和Cx43条件性基因敲除小鼠的连接蛋白半通道电流和缝隙连接通讯的多胺依赖性。具体目的2：确定不同类型的CXs在胶质瘤细胞中的过度表达如何参与细胞间的缝隙连接通讯，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目标之间的联系：连接蛋白和多胺。我们将定义多胺对不同CXs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CXS的调控，我们可以调节胶质瘤细胞之间的缝隙连接通讯，以及肿瘤微环境中胶质瘤细胞和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.