Role of autocrine motility factor in fibrosarcoma

1. gc - 4pf纤维肉瘤细胞AMF的纯化。我们之前已经证明，无蛋白培养的Gc-4 PF细胞与自分泌运动因子（AMF）的高反应及其受体gp78的高表达有关。通过连续的DEAE阴离子交换、CM阳离子交换和凝胶过滤层析，从培养基中纯化出两种具有离子交换特性的活性分子，即酸性和碱性AMFs。然而，两种AMFs在非还原和还原条件下的尺寸相似，分别为55kDa和65kDa， pI相同，均为6.5。百日咳毒素抑制了两种AMFs的运动，而透明质酸链霉菌酶则没有。这两种AMFs显著提高了自产gc - 4pf细胞的肺定殖特性1.5倍。这些结果表明，酸性和碱性AMFs都可能与先前报道的AMFs相对应。AMF-gp78信号的作用机理。Dunn骨肉瘤细胞经staurosporine和染料木素预孵生后，受刺激的运动性呈剂量依赖性和时间依赖性显著降低，而H-8对受刺激的运动性的影响可以忽略不计。这些数据表明，激酶C和酪氨酸激酶都在amf刺激的细胞运动中发挥作用，而蛋白激酶a可能不需要刺激。此外，一种运动抑制剂，侵袭抑制因子2 （IIF-2）抑制amf刺激的细胞运动。恶性肿瘤和类风湿关节炎（RA）患者AMF及其受体的表达。在118例结直肠癌患者中，gp78阳性的发生率与肿瘤进展相关，反映在组织学类型、淋巴结转移、血管侵犯和肿瘤分期上。提示gp78在结直肠癌患者中的表达可能具有预测预后的价值。我们证明了三个RA患者滑液的运动刺激活性。这种趋化动力学活性阻断单克隆抗体与AMF受体结合的能力在免疫印迹上得到证实。这些结果表明AMF在类风湿滑膜液中表达。目前尚未获得分泌抗AMF单克隆抗体的杂交瘤细胞。研究正在进行中。少

1. Purification of AMF derived from Gc-4 PF fibrosarcoma cells.We have previously demonstrated that protein-free culture of Gc-4 PF cells is associated with high response to autocrine motility factor (AMF) and with high expression of its receptor, gp78. Two motile activities, acidic and basic AMFs with regard to binding features on ion exchange chromatography, were purified from the cultured medium by sequential DEAE anion-exchange, CM cation-exchange, and gel-filtration chromatographies. However, the two AMFs have a similar size of 55kDa and 65kDa under nonreducing and reducing conditions, respectively, with same pI of 6.5. The stimulated motility of both AMFs was inhibited by the pertussis toxin, but not by Streptomyces hyaluronidase. These two AMFs significantly stimulated the lung colonizing properties of the self-producing Gc-4 PF cells by 1.5-fold. These results suggest that both acidic and basic AMFs may correspond to the previously reported AMF.2.Mechanism of AMF-gp78 signal tr … More ansduction.preincubation of Dunn osteosarcoma cells with both staurosporine and genistein resulted in a significant decrease in the stimulated motility in dose- and time-dependent manners, while H-8 had a negligible effect upon the stimulated motility. These data suggest that both kinase C and tyrosine kinase play a role in AMF-stimulated cell motility, while protein kinase A may not be required for the stimulation. Furthermore, a motility inhibitor, invasion-inhibiting factor 2 (IIF-2) suppressed the cell motility stimulated by AMF.3.Expression of AMF and its receptor in patients with malignant tumor and rheumatoid arthritis (RA).Among 118 patients with colorectal cancer, the incidence of gp78 positivity was correlated with tumor progression as reflected by histologic type, lymph node metastasis, vessel invasion, and tumor stage. The results indicate the expression of gp78 may be of prognostic value in patients with colorectal cancer.We demonstrated the motility stimulating activity in the synovial fluid from three patients with RA.The ability of this chemokinetic activity to block binding of the monoclonal antibody to the receptor for AMF was demonstrated on immunoblots. These results suggest that AMF is expressed in rheumatoid synovial fluids.4.Perspective.So far we could not obtain the hybridoma cells secreting monoclonal antibody against AMF.The studies are in progress now. Less