Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

Abstract

Summary Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anticatalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid.

Detection of in vivo enzyme activity with CatalyCEST MRI

“…For example, uPA can cleave a ZGGR peptidyl ligand attached to a 7‐amino‐4‐methylcoumarin (AMC), which causes the AMC to become highly fluorescent (Fig. a) . In addition, a second “control” fluorescent agent that is unresponsive to uPA activity can be selectively detected at the same time and in the same tissue location as the uPA‐responsive agent.…”

An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis

“…This peptide is a substrate for Urokinase-Type Plasminogen Activator (uPA), and the ability of the peptide-DOTA conjugate to detect uPA is the focus of ongoing research studies in our laboratory 31. Compound 7 was treated with Fmoc-Arg(pbf)-OH/HBTU/HOBt/DIEA for 1 hour and washed with NMP.…”