ROLE OF 02 RADICALS AND IRON IN ASBESTOS-INDUCED CANCER

02 自由基和铁在石棉诱发的癌症中的作用

• 批准号: 6382109
• 负责人: ANN E AUST
• 金额: $ 19万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382109
• 关键词: SDS polyacrylamide gel electrophoresis; asbestos; autocrine; biological signal transduction; chemical carcinogenesis; environment related neoplasm /cancer; enzyme activity; enzyme biosynthesis; free radical oxygen; genetic transcription; glutathione; human tissue; integrins; iron; ligands; messenger RNA; nitric oxide; nitric oxide synthase; nucleic acid biosynthesis; phosphorylation; receptor binding; tissue /cell culture; transfection /expression vector; transforming growth factors

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.

本研究的长期目标是探讨其机制