INDUCTION OF NF-KB AFTER EXPOSURE OF LYMPHOID CELLS TO SOLUBLE TAX1

淋巴细胞暴露于可溶性 TAX1 后诱导 NF-KB

• 批准号: 3853586
• 负责人: J BRADY
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3853586
• 关键词: DNA binding protein; cell nucleus; chromatography; gene expression; gene induction /repression; growth media; human T cell lymphotropic virus type 1; lymphocyte proliferation; phorbols; phosphoproteins; protein purification; radioimmunoassay; tissue /cell culture; transcription factor; transforming virus; western blottings

Purified HTLV-I Tax1 protein can be taken up by 70Z/3 lymphoid cells and localized in both the nuclear and cytoplasmic compartments. Introduction of the Tax1 protein into the growth medium of 7OZ/3 cells resulted in the rapid and transient induction of NF-KB binding activity in the nuclear fraction. Taxl activation of NF-kB was not sensitive to either staurosporin or prolonged stimulation with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate, suggesting that Taxl-dependent NF-kB activation did not require the protein kinase C pathway. Purified Taxl did not directly increase NF-KB binding activity in 7OZ/3 cytoplasmic extracts, suggesting that NF-KB induction may require cellular factors. Western blot and competitive radioimmunoassays demonstrated that Taxl protein was present in the tissue culture media of HTLV-I-transformed cell lines. These results show that extracellular Taxl may regulate cellular gene expression in noninfected cells. A purification protocol which involves sequential ammonium sulfate precipitation and zinc chelate chromatography to purify the HTLV-I Taxl protein expressed in E. coli has been developed. The final Taxl product is greater than 90% pure and the yield is approximately 1 mg per liter of liquid culture. The purified Tax protein is biologically active in indirect in vitro DNA binding assays, cellular NF-KB induction experiments and lymphocyte proliferation assays.

纯化的HTLV-I Tax 1蛋白可被70 Z/3淋巴样细胞摄取， 位于细胞核和细胞质区室中。介绍 在7 OZ/3细胞的生长培养基中加入Tax 1蛋白， 核内NF-κ B结合活性的快速和瞬时诱导 分数。 Taxl对NF-κ B的激活作用对两种药物均不敏感， 星形孢菌素或用佛波醇酯长时间刺激 12-0-十四酰基佛波醇-13-乙酸酯，表明紫杉醇依赖性 NF-κ B的激活不需要蛋白激酶C通路。 纯化 Taxl不直接增加7 OZ/3细胞中NF-κ B结合活性， 细胞质提取物，表明NF-κ B诱导可能需要 细胞因子 蛋白质印迹和竞争性放射免疫测定 证明Taxl蛋白存在于 HTLV-I转化的细胞系。 这些结果表明， Taxl可调节未感染细胞的细胞基因表达。 一种纯化方案，包括连续的硫酸铵 沉淀和锌螯合层析以纯化HTLV-I Taxl 在E.大肠杆菌已经开发。 Taxl最终产品 纯度大于90%，产率约为每升1毫克 液体培养 纯化的Tax蛋白具有生物活性， 间接体外DNA结合试验，细胞NF-κ B诱导 实验和淋巴细胞增殖测定。