ROLE OF 02 RADICALS AND IRON IN ASBESTOS-INDUCED CANCER

这项研究的长期目标是研究其机制。 通过这些活性氧物种，铁、谷胱甘肽和一氧化氮 会导致阿斯贝斯顿诱发的癌症。建议的假设 研究表明，石棉可与整合素受体结合，从而激活 刺激谷胱甘肽外流和降低细胞周期的磷酸化信号转导途径 谷氨酰半胱氨酸合成酶(GCS)的活性及其速率控制 GSH合成中的酶，导致诱导型一氧化氮的产生 一氧化氮合酶(INOS)和一氧化氮的产生。具体目标是：1)确定 整合素受体的激活是否导致GSH外流和 测定谷氨酰胺半胱氨酸合成酶活性；2)测定 转化生长因子-β1是青石棉诱导的血管内皮细胞减少的自分泌介质 细胞内GSH；3)研究暴露后GCS活性如何降低 青石棉；以及4)研究细胞内铁和a的减少 谷胱甘肽可引起Src激酶的激活。将使用人类肺上皮细胞 因为它们是石棉诱发癌症的靶细胞 因为诱导型一氧化氮合酶在人类体内的诱导似乎受到不同的调控 比在其他物种中更多。特异性整合素抗体和特异性整合素抗体 结合多肽将被用来确定存在哪些整合素和 整合素配体的结合，如青石棉、玻连素和 HIV-Tat蛋白，导致GSH外流刺激和GCS下降 活动。接下来，将确定转化生长因子-β1的产生是否 导致自分泌刺激，导致细胞内 谷胱甘肽。它是否以活动形式生产，将通过使用 荧光素酶报告细胞。αvβ6整合素是否参与 激活将使用针对此的阻断抗体来确定 整合素。对转化生长因子β1的诱导合成将通过以下方式确定 测定mRNA的合成。还将确定是否治疗 含有转化生长因子-β1的细胞，在没有整合素结合配体的情况下会导致 GCS转录减少。如果是，则包含 将GCS重亚单位导入细胞，观察是否诱导 Inos是可以逆转的。以确定各种整合素结合配体是否 降低GCS活性，对细胞进行配体和GCS处理 活动将被确定。确定这些影响的调解是否会对 GCS的活性是通过cAMP和蛋白激酶C，特异性抑制和 将使用刺激性试剂。为了确定铁和谷胱甘肽是如何调节 调节半胱氨酸的单溴莫比胺标记的Src激酶活性将 被监视。

The long-term objective of this research is to investigate the mechanism by which reactive oxygen species, iron, glutathione (GSH) and NO contribute to asbeston-induced cancer. The hypothesis for the proposed research is that asbestos binds integrin receptors leading to activation of phosphorylation signaling pathways which stimulate GSH efflux and decrease the activity of gamma-glutamylcysteine synthetase (GCS), the rate-controlling enzyme in GSH synthesis, leading to induction of the inducible form of nitric oxide synthase (iNOS and NO production. The specific aims are: 1) to determine whether activation of integrin receptors leads to GSH efflux and a decrease in the activity of gamma-glutamycysteine synthetase; 2) to determine whether TGF-beta 1 is an autocrine mediator of crocidolite induced decrease in intracellular GSH; 3) to investigate how GCS activity decreases after exposure to crocidolite; and 4) to investigate how iron and a decrease in intracellular GSH cause activation of Src kinase. Human lung epithelial cells will be used for these studies because they are the target cells for asbestos-induced cancer and because the induction of iNOS in humans appears to be regulated differently than in other species. Antibodies to specific integrins and specific integrin binding peptides will be used to determine which integrins are present and whether binding of integrin ligands, such as crocidolite, vitronectin and HIV-Tat protein, leads to a stimulation in GSH efflux and a decrease in GCS activity. Next, it will be determined whether production of TGF-beta 1 is resulting in autocrine stimulation that leads to a decrease in intracellular GSH. Whether it is being produced in an active form will be determined by using luciferase reporter cells. Whether the alpha v beta 6 integrin is involved in the activation will be determined using blocking antibodies specific for this integrin. The induction of synthesis of TGF beta 1 will be determined by measuring synthesis of mRNA. It will also be determined whether treatment of cells with TGF-beta 1, in the absence of integrin-binding ligands will lead to a decrease in transcription of GCS. If so, an expression vector containing the GCS heavy subunit will be transfected into the cells to see if the induction of iNOS can be reversed. To determine whether various integrin-binding ligands decrease the activity of GCS, cells will be treated with the ligands and GCS activity will be determined. To determine whether mediation of these effects on GCS activity are through cAMP and protein kinase C, specific inhibitory and stimulatory reagents will be used. To determine how iron and GSH modulate the activity of Src kinase, monobromobimane labeling of regulatory cysteines will be monitored.