| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
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
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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
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