Rusnati M, Coltrini D, Campioni D, Tanghetti E, Corallini A, Barbanti-Brodano G, Giuliani R, Gibellini D, Presta M
Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy.
AIDS. 1997 May;11(6):727-36. doi: 10.1097/00002030-199706000-00005.
To demonstrate that Tat modulates the plasminogen-dependent proteolytic activity of tumour cell lines derived from BK virus (BKV)/tat-transgenic mice by affecting the production of plasminogen activators (PA) and the PA inhibitor (PAI)-1 and to demonstrate that this occurs through mechanism(s) that are distinct from those responsible for transactivating activity of extracellular Tat.
To assess whether endogenous Tat is responsible for PA activity in T53 adenocarcinoma cells, cell cultures were transfected with antisense Tat cDNA and evaluated for cell-associated PA activity by a plasmin chromogenic assay. The assay was also used to evaluate PA activity in T53 cells and T111 leiomyosarcoma cells stimulated by extracellular Tat. The type(s) of PA produced were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. The levels of PAI-1 were evaluated by Western blotting. Tat transactivating activity was measured by a chloramphenicol acetyltransferase (CAT) enzyme-linked immunosorbent assay in HL3T1 cells containing integrated copies of an HIV-1 long terminal repeat (LTR)-CAT plasmid.
Transfection of T53 cells with antisense Tat cDNA results in the decrease of Tat production and PA activity. Exogenously added Tat increases PA levels in T53 and in T111 cells. PA activity was identified as urokinase-type PA (uPA). Tat also increases the production of PAI-1 in T111 but not in T53 cells. Chloroquine and heparin have different affects on the LTR-CAT-transactivating and the PA-inducing activities of Tat. The fusion protein glutathione-S-transferase-Tat and the mutant Tat-1e, lacking the second Tat exon, cause LTR-CAT transactivation without stimulating uPA upregulation.
Tat affects the fibrinolytic activity of tumour cell lines derived from BKV/tat-transgenic mice by modulating the production of both uPA and PAI-1 via autocrine and paracrine mechanisms of action. The capacity of Tat to modulate the plasminogen-dependent proteolytic activity of these tumour cell lines may contribute to their metastatic potential. The uPA-inducing activity of Tat depends upon specific biological and structural features of the Tat protein that are distinct from those responsible for its LTR-CAT-transactivating activity, suggesting distinct mechanisms of induction for the two biological responses.
证明Tat通过影响纤溶酶原激活剂（PA）和PA抑制剂（PAI）-1的产生，调节源自BK病毒（BKV）/tat转基因小鼠的肿瘤细胞系的纤溶酶原依赖性蛋白水解活性，并证明这是通过与负责细胞外Tat反式激活活性的机制不同的机制发生的。
为评估内源性Tat是否负责T53腺癌细胞中的PA活性，用反义Tat cDNA转染细胞培养物，并通过纤溶酶显色测定法评估细胞相关的PA活性。该测定法还用于评估细胞外Tat刺激的T53细胞和T111平滑肌肉瘤细胞中的PA活性。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳酶谱法鉴定产生的PA类型。通过蛋白质印迹法评估PAI-1的水平。在含有HIV-1长末端重复序列（LTR）-氯霉素乙酰转移酶（CAT）质粒整合拷贝的HL3T1细胞中，通过CAT酶联免疫吸附测定法测量Tat反式激活活性。
用反义Tat cDNA转染T53细胞导致Tat产生和PA活性降低。外源添加的Tat增加T53和T111细胞中的PA水平。PA活性被鉴定为尿激酶型PA（uPA）。Tat还增加T111细胞中PAI-1的产生，但不增加T53细胞中PAI-1的产生。氯喹和肝素对Tat的LTR-CAT反式激活和PA诱导活性有不同影响。融合蛋白谷胱甘肽-S-转移酶-Tat和缺乏第二个Tat外显子的突变体Tat-1e引起LTR-CAT反式激活而不刺激uPA上调。
Tat通过自分泌和旁分泌作用机制调节uPA和PAI-1的产生，从而影响源自BKV/tat转基因小鼠的肿瘤细胞系的纤溶活性。Tat调节这些肿瘤细胞系的纤溶酶原依赖性蛋白水解活性的能力可能有助于其转移潜能。Tat的uPA诱导活性取决于Tat蛋白的特定生物学和结构特征，这些特征不同于负责其LTR-CAT反式激活活性的特征，表明这两种生物学反应的诱导机制不同。
