CELL ADHESION, EXTRACELLULAR FACTORS & RADIATION ARREST

细胞粘附、细胞外因子

• 批准号: 2909858
• 负责人: DONNA M GADBOIS
• 金额: $ 21.51万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2909858
• 关键词: DNA replication; actins; binding proteins; cell adhesion; cell cycle; cell differentiation; cyclin dependent kinase; enzyme inhibitors; extracellular matrix proteins; fibroblasts; fibronectins; free radical oxygen; gene expression; human tissue; immunofluorescence technique; integrins; laminin; neutralizing antibody; oncoprotein p21; p53 gene /protein; protein protein interaction; protein purification; radiation genetics; receptor binding; transforming growth factors

It is thought that radiation causes a p53-dependent G1 phase delay in normal cells to allow additional time for repair of DNA damage. However, it has been reported that human fibroblasts, unlike other cell types, do not delay after irradiation, but arrest permanently in G1 phase with a senescence-like phenotype. We have shown that: 1) the "permanent" arrest is, in fact, reversible in a substantial population of the irradiated culture if adhesion interactions are disrupted; 2) the arrest is dependent on the type of extracellular matrix (ECM); 3) the maintenance of the arrest depends on an extracellular factor(s); and 4) arrest results in the expression of smooth muscle alpha-actin, characteristic of the differentiated myofibroblast. We propose that candidate extracellular factors are reactive oxygen species (ROS) and TGF-beta1. We suggest that radiation causes an increase in ROS, which activates TGF-beta1. Cells might then arrest because of the direct action of TGF-beta1, or through the binding of RGD sequences in latency associated peptide (LAP) or latent TGF-beta1 binding protein (LTBP) to growth inhibitory integrins. Fibroblasts might differentiate to myofibroblasts because of TGF-beta1 induction of ED-A fibronectin, a spliced form of fibronectin that has been shown to cause differentiation of fibroblasts in response to TGF-beta1. The ECM dependency of arrest could result from a preferential activation, expression, or localization of these proteins. These studies are important because the effect of the extracellular environment on radiation arrest is rarely examined. Also, these studies are directly relevant to, and will have therapeutic impact on the treatment of radiation fibrosis because they focus on how the production and interplay of ROS and TGF-beta1 affect radiation cell cycle arrest and differentiation of fibroblasts in response to ECM signals. For example, TGF-beta1-induced myofibroblast differentiation is thought to play an important role in radiation fibrosis. In fact, clinical studies show that increased amounts of TGF-beta1 and the presence of myofibroblasts are an indicator of fibrotic progression. In the first year of the proposed work, we will study the interactions of ROS and TGF-beta1, and begin experiments with TGF-beta1, LAP, and LTBP-1 measurements and immunolocalization. In the second year, we will conclude work on TGF-beta1, LAP, and LTBP-1 measurements and immunolocalization, and perform experiments concerning ED-A fibronectin. In year three, we will measure amounts and kinase activities of CDKs and CKIs under conditions of radiation arrest, or ROS or TGF-beta1 inhibition. In the fourth year, we will identify growth inhibitory integrin receptors that interact with LTBP-1 or LAP after irradiation.

据认为，辐射导致正常细胞中依赖于P53的G1期延迟，从而为DNA损伤的修复提供了额外的时间。然而，据报道，与其他类型的细胞不同，人成纤维细胞在照射后不会延迟，而是永久停滞在具有衰老样表型的G1期。我们已经证明：1)事实上，在大量受照射的培养细胞中，如果黏附相互作用被破坏，“永久性”停滞是可逆的；2)停滞取决于细胞外基质的类型；3)停滞的维持依赖于细胞外因子(S)；以及4)停滞导致平滑肌α-肌动蛋白的表达，这是分化的肌成纤维细胞的特征。我们认为候选的细胞外因子是活性氧(ROS)和转化生长因子-β1。我们认为，辐射导致ROS增加，从而激活转化生长因子-β1。然后，细胞可能因为转化生长因子-β1的直接作用，或通过潜伏期相关肽(LAP)或潜在的转化生长因子-β1结合蛋白(LTBP)中的RGD序列与生长抑制整合素的结合而停止生长。成纤维细胞可能分化为肌成纤维细胞，因为转化生长因子-β1诱导ED-A纤维连接蛋白，这是纤维连接蛋白的一种拼接形式，已被证明引起成纤维细胞的分化，以响应转化生长因子-β1。这些蛋白的优先激活、表达或定位可能导致细胞外基质对细胞外基质的依赖。这些研究很重要，因为很少有人研究细胞外环境对辐射停滞的影响。此外，这些研究与放射性纤维化的治疗直接相关，并将对其产生治疗影响，因为它们侧重于ROS和转化生长因子-β1的产生和相互作用如何影响辐射细胞周期停滞和成纤维细胞对ECM信号的反应分化。例如，转化生长因子-β1诱导的肌成纤维细胞分化被认为在放射性纤维化中起重要作用。事实上，临床研究表明，转化生长因子-β1的增加和肌成纤维细胞的存在是纤维化进展的一个指标。在拟议工作的第一年，我们将研究ROS和转化生长因子-β1的相互作用，并开始实验转化生长因子-β1、LAP和LTBP-1的测量和免疫定位。在第二年，我们将完成关于转化生长因子-β1、LAP和LTBP-1的测量和免疫定位的工作，并进行关于ED-A纤维连接蛋白的实验。在第三年，我们将测量CDK和CKI在辐射停止或ROS或转化生长因子-β1抑制条件下的数量和激酶活性。在第四年，我们将确定在照射后与LTBP-1或LAP相互作用的生长抑制整合素受体。