Production of pancreatic secretory trypsin inhibitor (PSTI) as a growthstimulating factor and characterization of its specific receptors

作为生长刺激因子的胰腺分泌性胰蛋白酶抑制剂（PSTI）的生产及其特异性受体的表征

基本信息

批准号：
63480135
负责人：
OGAWA Michio
金额：
$ 2.18万
依托单位：
Osaka University Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

Various cancer tissues contained pancreatic secretory trypsin inhibitor ( PSTI )-immunoreactive cells. Especially almost all adenocarcinorna tissues contained PSTI-immunoreactive cells, though the proportion of PSTI-positive cells varied in degree. Cultured human cells in protein-free nutrient medium secreted a considerable amount of PSTI into culture medium. The production of PSTI by cancer cells was confirmed by the isolation of PSTI cDNA clones from neoplastic tissues, the nucleotide sequence of neoplastic tissue PSTI cDNA being completely identical with that of pancreatic PSTI cDNA. Specific binding of ^<125>I-PSTI was noted with the following cells; WI-38, 3T3 Swiss albino, HUVE, BDC-1 and H4-lI-E-C3. Specific binding sites or human PSTI on 3T3 Swiss albino cells were studied using radioiodinated recombinant PSTI. On Scatchard analysis of the competitive binding data, linear plots indicated a single class of receptors with high affinity( Kd - 5.3 x 10^<-10>M ) on 3T3 Swiss albino … More cells, the number of receptors being 5,400 per cell. Treatment of surface bound radiolabeled PSTI with a chemical crosslinker ( disuccinimidyl suberate ) led to the identification of a membrane polypeptide of Mr 140,000 to which PSTI was crosslinked. The formation was inhibited by an excess amount of unlabeled PSTI in a dose dependent manner. The binding of ^<125>I-PSTI to 313 Swiss albino cells was competitively inhibited by unlabeled PSTI but not by other peptide hormones, such as EGF, b-FGF, IGF-I, TGF-alpha, PDGF and TNF, indicating the presence of receptors specific for PSTI. Various protease inhibitors had no or only a little effect, and mercaptoethanol and dithiothreitol strongly decreased the binding of ^<125>I-PSTI. Incubation at 37ﾟC resulted in rapid internalization of cell bound ^<125>I-PSTI, followed by the appearance of trichloroacetic acid-soluble ^<125>I-radioactivity in the culture medium, due to degradation of PSIT. In addition, PSTI stimulated [3^H]thymidine incorporation into DNA on 313 Swiss albino cells in a dose dependent manner. These results indicated that the biological effect of PSIT was mediated by high affinity plasma membrane receptors, which were not a cell-surface proteinase(s). Less

各种癌症组织包含胰腺秘密胰蛋白酶抑制剂（PSTI） - 免疫反应性细胞。尤其是几乎所有腺癌组织都包含PSTI免疫反应性细胞，尽管PSTI阳性细胞的比例在程度上有所不同。无蛋白质养分培养基中培养的人类细胞将考虑数量的PSTI分泌为培养基。通过从肿瘤组织中分离PSTI cDNA克隆来证实癌细胞的PSTI，肿瘤组织PSTI cDNA的核肽序列与胰腺PSTI cDNA完全相同。 ^<125> i-pSTI的特异性结合在以下细胞中被注意。 WI-38，3T3瑞士白化病，HUVE，BDC-1和H4-LI-E-C3。使用放射性固生重组PSTI研究了3T3瑞士白化病细胞上的特定结合位点或人类PSTI。在对竞争性结合数据的SCATCHARD分析时，线性图表明，在3T3 Swiss Albino上具有高亲和力（KD -5.3 x 10^<-10> M）的单一类受体…更多的细胞，更多的细胞，每个细胞的受体数量为5,400。用化学交叉链链链链链链链链链接（二甲酰亚胺基）处理表面结合的放射性标记的PSTI，导致鉴定了MR 140,000的膜多肽，PSTI是140,000的。超过 ^<125> I-PSTI与313个瑞士白化病细胞的结合抑制了形成。未标记的PSTI竞争抑制了PSTI，但没有其他肽恐怖剂，例如EGF，B-FGF，IGF-I，TGF-A，TGF-Alpha，tgf-alpha，PDGF和TNF，以表明受体的特定于PSTI。各种蛋白质抑制剂没有或仅有一点效果，硫乙醇和二硫代醇可强烈降低 ^<125> i-PSTI的结合。在37°C处的孵育导致了结合 ^<125> i-PSTI的细胞快速内在化，然后出现三氯乙酸 - 溶解 ^<125> i-radioactivity在培养基中，由于PSIT的降解。此外，PSTI以剂量依赖性方式刺激了[3^H]胸苷在313个瑞士白化病细胞上掺入DNA。这些结果表明，PSIT的生物学效应是由高亲和力质膜受体介导的，这些质膜受体不是细胞表面蛋白酶（S）。较少的