STIMULATED MOTILITY IN TUMOR CELLS

刺激肿瘤细胞的运动能力

• 批准号: 6290728
• 负责人: Lance Allen Liotta
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 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/6290728
• 关键词: adenosine; adenosine monophosphate; adenosine triphosphate; cell communication molecule; cell motility; cellular oncology; enzyme activity; human tissue; metastasis; molecular cloning; phosphodiesterase I; purinergic receptor; site directed mutagenesis; tissue /cell culture

We have been studying tumor cell motility as a component of the process of metastasis with a special emphasis on factors that could regulate this motility. To this end, we purified, cloned, and sequenced autotaxin, a tumor-secreted motility-stimulating glycoprotein from the conditioned medium of a human melanoma cell line. Utilizing reverse transciption followed by PCR, we have also isolated autotaxin from human breast carcinoma and teratocarcinoma cell lines. Northern blot analysis of multiple normal tissues indicated that this protein has the highest steady state levels in ovary, small intestine, brain and placenta. In addition to its motility-stimulating properties, autotaxin possesses type I phosphodiesterase, ATPase, nucleotide pyrophosphatase, inorganic pyrophosphatase, 5|-nucleotidase, autophosphorylation, and ATP binding activities. Recombinant autotaxin, purified to homogeneity, retains all of the above activities and stimulates motility at high picomolar to low nanomolar concentrations. Though the human melanoma cell line, A2058, has been used as the motility model in most of the purifications of autotaxin, we have found that certain breast and prostatic carcinoma cells, neuroblastoma cells, bovine aortic smooth muscle cells, as well as HUVEC|s (human umbilical vein cells) also respond to autotaxin as a chemoattractant. Studies utilizing site- directed mutagenesis of the phosphodiesterase active site demonstrated that an intact phosphodiesterase catalytic site is necessary for motility, but phosphorylation of the molecule does not appear to be a prerequisite. Since autotaxin has no known ATP binding site, we utilized a radiolabelled 8-azido-ATP to demonstrate ATP binding to autotaxin and to localize two regions on the molecule where the 8- azido-ATP crosslinks after exposure to light. The first region is the phosphodiesterase active site itself. The second region is 108 amino acids downstream, is unlike any reported ATP binding sites, and is sensitive to addition of salt. We have found that autotaxin possesses type I phosphodiesterase, ATPase, nucleotide pyrophosphatase, autophosphorylation, and ATP binding activities. Utilizing site- directed mutagenesis, we explored the relationship between the enzymatic properties of autotaxin and its capacity to stimulate motility. We found that a single point mutation in the phosphodiesterase catalytic site (Thr210 Ala or Asp) inhibited its phosphodiesterase, autophosphorylation, and motility-stimlating activities but had no effect on ATP binding. A second point mutation (Lys209 Leu) inhibited autophosphorylation but not motility stimulation or phosphodiesterase. Therefore, an intact phosphodiesterase catalytic site is necessary for motility, but phosphorylation of the molecule does not appear to be a prerequisite. Finally, we have found that AMP and adenosine are chemoattractants for the human melanoma cell line, A2058. These molecules, which are products of autotaxins interaction with ATP, appear to act through an A1 adenosine receptor. Transfection of autotaxin into 3T3 cells which have an activated ras oncogene profoundly stimulates the invasive, tumorigenic and metastatic properties of these cells, compared to controls.

我们一直在研究肿瘤细胞运动，是转移过程的一个组成部分，特别强调可能调节这种运动的因素。为此，我们从人黑色素瘤细胞系的条件培养基中纯化，克隆和测序的自身Xt蛋白，一种刺激糖蛋白的肿瘤分泌性糖蛋白。利用反向转递，然后是PCR，我们还从人类乳腺癌和Teratocarcinoma细胞系中分离了自身X月。对多个正常组织的北印迹分析表明，该蛋白在卵巢，小肠，大脑和胎盘中具有最高的稳态水平。除了刺激运动性能外，自身X.蛋白还具有I型磷酸二酯酶，ATPase，核苷酸焦磷酸酶，无机焦磷酸酶，5 | - 核苷酸酶，自磷酸化和ATP结合活性。重组自身蛋白酶（纯化为均匀性）保留上述所有活动，并在高皮摩尔至低纳摩尔浓度下刺激运动性。尽管人类黑色素瘤细胞系A2058在大多数自身Xt蛋白的纯化中已被用作运动模型，但我们发现某些乳腺癌和前列腺癌细胞，神经母细胞瘤细胞，牛主动脉平滑肌细胞，Huvec | s（人体静脉细胞）也对自动抗体抗体反应。利用磷酸二酯酶活性位点的位置定向诱变的研究表明，完整的磷酸二酯酶催化位点对于运动性是必要的，但是分子的磷酸化似乎不是先决条件。由于自动脱霉素没有已知的ATP结合位点，因此我们利用了放射性标记的8-齐达多-ATP来证明ATP与自身X。第一个区域是磷酸二酯酶活性位点本身。第二个区域是下游的108个氨基酸，与任何报道的ATP结合位点不同，并且对添加盐敏感。我们发现自身酰胺具有I型磷酸二酯酶，ATPase，核苷酸焦磷酸酶，自磷酸化和ATP结合活性。利用位置定向的诱变，我们探索了自动昔候酶特性与其刺激运动能力之间的关系。我们发现，磷酸二酯酶催化位点（THR210 ALA或ASP）中的单点突变抑制其磷酸二酯酶，自磷酸化和刺激性刺激活性，但对ATP结合没有影响。第二点突变（LYS209 LEU）抑制了自磷酸化，而不是运动刺激或磷酸二酯酶。因此，完整的磷酸二酯酶催化位点对于运动性是必要的，但是分子的磷酸化似乎不是先决条件。最后，我们发现AMP和腺苷是人类黑色素瘤细胞系A2058的趋化剂。这些分子是与ATP相互作用的产物，似乎通过A1腺苷受体起作用。与对照组相比，将自身X线转染到具有活化的Ras癌基的3T3细胞中，这些细胞深刻刺激了这些细胞的侵入性，肿瘤和转移性特性。