Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line

Kei Takahashi*, Takashi Gojobori, Masaaki Tanifuji

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10-13 to 10-11 mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate.

Original languageEnglish (US)
Pages (from-to)405-413
Number of pages9
JournalExperimental Cell Research
Volume192
Issue number2
DOIs
StatePublished - Jan 1 1991

ASJC Scopus subject areas

  • Cell Biology

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