CELL ADHESION, EXTRACELLULAR FACTORS & RADIATION ARREST

细胞粘附、细胞外因子

• 批准号: 6513423
• 负责人: DONNA M GADBOIS
• 金额: $ 27.02万
• 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 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6513423
• 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的p53依赖性G1期延迟，以允许额外的时间修复DNA损伤。 然而，据报道，人类成纤维细胞，不像其他类型的细胞，不延迟照射后，但永久停滞在G1期与衰老样表型。我们已经证明：1）如果粘附相互作用被破坏，则“永久”停滞实际上在照射培养物的大量群体中是可逆的; 2）停滞依赖于细胞外基质（ECM）的类型; 3）停滞的维持依赖于细胞外因子;和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。我们建议，候选细胞外因子是活性氧（ROS）和TGF-β 1。 我们认为，辐射会导致ROS增加，从而激活TGF-β 1。 然后，细胞可能由于TGF-β 1的直接作用而停滞，或者通过潜伏相关肽（LTP）或潜伏TGF-β 1结合蛋白（LTBP）中的RGD序列与生长抑制性整联蛋白的结合而停滞。 成纤维细胞可能分化成肌成纤维细胞，因为TGF-β 1诱导ED-A纤连蛋白，一种剪接形式的纤连蛋白，已显示引起成纤维细胞响应TGF-β 1的分化。细胞外基质依赖性的停滞可能是由于这些蛋白质的优先激活、表达或定位。这些研究很重要，因为细胞外环境对辐射阻滞的影响很少被研究。此外，这些研究与放射性纤维化的治疗直接相关，并将对放射性纤维化的治疗产生治疗影响，因为它们关注ROS和TGF-β 1的产生和相互作用如何影响辐射细胞周期阻滞和成纤维细胞响应ECM信号的分化。 例如，TGF-β 1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，TGF-β 1的增加和肌成纤维细胞的存在是纤维化进展的指标。在拟议工作的第一年，我们将研究活性氧和TGF-β 1的相互作用，并开始实验与TGF-β 1，TGF-β 1和LTBP-1的测量和免疫定位。第二年完成TGF-β 1、TGF β 1、LTBP-1的测定和免疫定位，并进行ED-A纤连蛋白的实验。 在第三年，我们将测量在辐射停滞或ROS或TGF-β 1抑制条件下CDKs和CKIs的数量和激酶活性。在第四年，我们将确定生长抑制整合素受体与LTBP-1或照射后相互作用。