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

多种癌组织中含有胰腺分泌性胰蛋白酶抑制剂（PSTI）免疫反应细胞。特别是几乎所有的腺癌组织中都含有psti免疫反应细胞，尽管psti阳性细胞的比例不同程度。在无蛋白营养培养基中培养的人细胞向培养基中分泌了相当数量的PSTI。从肿瘤组织中分离到PSTI cDNA克隆，证实肿瘤细胞产生PSTI，肿瘤组织PSTI cDNA的核苷酸序列与胰腺PSTI cDNA完全一致。在以下细胞中观察到^<125>I-PSTI的特异性结合；WI-38, 3T3瑞士白化，HUVE， BDC-1和H4-lI-E-C3。用放射性碘化重组PSTI研究了人PSTI在3T3瑞士白化细胞上的特异性结合位点。通过对竞争结合数据的Scatchard分析，线性图显示在3T3瑞士白化病细胞上存在一类高亲和力受体（Kd - 5.3 x 10^<-10>M），每个细胞有5400个受体。用化学交联剂（亚酸二琥珀酰）处理表面结合的放射性标记的PSTI，鉴定出了一个Mr为140,000的膜多肽，PSTI与之交联。过量未标记的PSTI以剂量依赖的方式抑制其形成。^<125 bb0 I-PSTI与313个瑞士白化细胞的结合被未标记的PSTI竞争性地抑制，但不被其他肽激素（如EGF、b-FGF、IGF-I、tgf - α、PDGF和TNF）抑制，表明存在PSTI特异性受体。多种蛋白酶抑制剂均无或仅有少量作用，巯基乙醇和二硫苏糖醇可显著降低^<125 bb0 I-PSTI的结合。在37℃的孵育下，由于PSIT的降解，细胞结合的^<125>I-PSTI迅速内化，随后在培养基中出现三氯乙酸溶解的^<125> i -放射性。此外，PSTI以剂量依赖的方式刺激313个瑞士白化病细胞的[3^H]胸苷结合到DNA中。这些结果表明PSIT的生物学效应是由高亲和力的质膜受体介导的，而不是细胞表面蛋白酶。少

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