The Src Kinase, cell communication & growth control

描述（申请人提供）：肿瘤细胞的生长可以被周围的正常细胞控制。这个过程被称为“异源生长控制”。这个项目的目标是阐明这个过程背后的机制。我们将确定未转化细胞如何影响邻近转化细胞中的Src信号级联。异种生长控制中异种间隙连接通讯的必要性将在特异性目标1中进行评估。我们假设非转化细胞不需要与Src转化细胞形成活跃的间隙连接通道来使其正常生长。我们将利用连接蛋白敲除细胞、化学阻滞剂或反义核酸来抑制间隙连接通讯。细胞间通讯将通过荧光染料、内源性代谢物和电导的转移来检查。如果异源生长控制发生在没有间隙连接通信的情况下，那么，正如我们假设的那样，异源生长控制不需要间隙连接通信。非转化细胞对Src激酶活性的影响，以及Src转化细胞中Src下游激酶活性的影响将分别在Specific Aims 2和3中进行评估。我们假设未转化的细胞抑制Src激酶活性，并影响邻近转化细胞中下游激酶的活性。我们将使用我们开发的新技术来检查转化细胞中的激酶活性，这些细胞在形态学上被邻近的非转化细胞逆转为正常表型。如果转化细胞级联中的Src信号被与非转化细胞的通信阻断，我们的假设将得到证实。非转化细胞对邻近转化细胞中基因整体表达模式的影响将在Specific Aim 4中进行研究。我们假设未转化的细胞改变了邻近转化细胞中特定基因的表达。我们将利用DNA芯片来比较转化细胞与非转化细胞共培养、转化细胞和单独培养的非转化细胞的基因谱。如果在异源生长控制过程中，转化细胞中某些基因的表达水平受到与非转化细胞接触的影响，而其他基因的表达水平则不受影响，那么这一假设将得到证实。

DESCRIPTION (provided by applicant): Tumor cell growth can be controlled by surrounding normal cells. This process is called "heterologous growth control". The goal of this project is to elucidate mechanisms underlying this process. We will determine how nontransformed cells affect the Src signaling cascade in neighboring transformed cells. The need for heterologous gap junctional communication in heterologous growth control will be evaluated in Specific Aim 1. We hypothesize that nontransformed cells do not need to form active gap junction channels with Src transformed cells to normalize their growth. We will utilize connexin knockout cells, chemical blockers or antisense nucleic acids to suppress gap junctional communication. Intercellular communication will be examined by the transfer of fluorescent dyes, endogenous metabolites, and electrical conductance. If heterologous growth control occurs in the absence of gap junctional communication, then, as we hypothesize, gap junctional communication is not required for heterologous growth control. Effects of nontransformed cells on the Src kinase activity, and the activity of kinases acting downstream of Src, in Src transformed cells will be evaluated in Specific Aims 2 and 3, respectively. We hypothesize that nontransformed cells quell Src kinase activity, and affect the activity of downstream kinases, in neighboring transformed cells. We will use novel techniques that we have developed to examine kinase activity in transformed cells that are morphologically reversed to a normal phenotype by neighboring nontransformed cells. Our hypothesis will be proven if the Src signaling in transformed cells cascade is blocked by communication with nontransformed cells. Effects of nontransformed cells on the global expression pattern of genes in neighboring transformed cells will be examined in Specific Aim 4. We hypothesize that nontransformed cells alter the expression of specific genes in neighboring transformed cells. We will utilize DNA chips to compare the gene profiles of transformed cells cocultured with nontransformed cells to transformed cells and nontransformed cells grown alone. The hypothesis will be proven if the expression levels of some genes, but not others, in transformed cells are affected by contact with nontransformed cells during heterologous growth control.