CELL ADHESION, EXTRACELLULAR FACTORS & RADIATION ARREST

据认为，辐射会导致正常细胞中p53依赖性的G1期延迟，从而有更多的时间来修复DNA损伤。然而，据报道，与其他细胞类型不同，人类成纤维细胞在照射后不会延迟，而是永久地停留在G1期，具有衰老样表型。我们已经证明：1)如果粘附相互作用被破坏，在辐照培养的大量人群中，“永久性”阻滞实际上是可逆的；2)阻滞取决于细胞外基质（ECM）的类型；3)逮捕的维持取决于细胞外因素；4)阻滞导致平滑肌α -肌动蛋白的表达，这是分化的肌成纤维细胞的特征。我们提出候选的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。由于TGF-beta1的直接作用，或者通过潜伏期相关肽（LAP）或潜伏TGF-beta1结合蛋白（LTBP）中的RGD序列与生长抑制整合素的结合，细胞可能会停滞。由于tgf - β a1诱导ED-A纤维连接蛋白，成纤维细胞可能分化为肌成纤维细胞，ED-A纤维连接蛋白是纤维连接蛋白的一种剪接形式，已被证明在tgf - β a1的作用下可导致成纤维细胞分化。ECM对阻滞的依赖性可能是由于这些蛋白的优先激活、表达或定位。这些研究很重要，因为细胞外环境对辐射抑制的影响很少被研究。此外，这些研究与放射性纤维化的治疗直接相关，并将对治疗产生影响，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对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辐射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一个指标。在提出的工作的第一年，我们将研究ROS和TGF-beta1的相互作用，并开始TGF-beta1， LAP和LTBP-1测量和免疫定位的实验。在第二年，我们将完成tgf - β 1， LAP和LTBP-1的测量和免疫定位的工作，并进行ED-A纤维连接蛋白的实验。在第三年，我们将测量辐射阻滞或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，我们将确定辐照后与LTBP-1或LAP相互作用的生长抑制整合素受体。

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.