Nierodzik M L, Chen K, Takeshita K, Li J J, Huang Y Q, Feng X S, D'Andrea M R, Andrade-Gordon P, Karpatkin S
New York University Medical Center and Kaplan Cancer Center, New York, NY, USA.
Blood. 1998 Nov 15;92(10):3694-700.
Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P <.04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P =.0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.
凝血酶处理的肿瘤细胞在实验性小鼠肺转移中诱导转移表型。凝血酶与一种独特的蛋白酶激活受体（PAR-1）结合，该受体需要通过其拴系末端进行N端蛋白水解切割才能激活。拴系末端的14聚体凝血酶受体激活肽（TRAP）诱导与凝血酶相同的细胞变化。通过逆转录聚合酶链反应（RT-PCR）检测，四种小鼠肿瘤细胞（Lewis肺癌、CT26结肠癌细胞、B16F10黑色素瘤细胞和CCL163成纤维细胞）含有PAR-1。B16F10细胞不含有另外两种凝血酶受体PAR-3和糖蛋白Ib。经TRAP处理的B16F10肿瘤细胞使肺转移增加41至48倍（n = 17）。用全长小鼠PAR-1正义cDNA（S6、S7、S14和S22）转染的经凝血酶处理的B16F10细胞使其与纤连蛋白的粘附增强1.5至2.4倍（n = 5，P <.04），而经凝血酶处理的野生型细胞则没有。通过RT-PCR和Northern分析检测时，S6（粘附指数，1.5倍）和S14（指数，2.4倍）显示S6相对于野生型PAR-1表达极少，而S14表达相当。免疫组织化学显示，与野生型或空质粒转染细胞相比，S14的PAR-1表达更高。对经凝血酶处理的S14转染体进行的体内实验显示，与空质粒转染的经凝血酶处理的未处理细胞或S6细胞相比，转移增加了五至六倍（n = 20，P =.0001至.02）。反义核酸对凝血酶刺激的肿瘤块没有影响。因此，PAR-1的连接和表达增强并调节肿瘤转移。
