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Determination of the Levels of Urokinase and Its Receptor in Human Colon Carcinoma Cell Lines¹

Department of Pharmacology, Bristol-Baylor Laboratory, Baylor College of Medicine, Houston, Texas 77030

At present, there is a lack of availability of differentiation markers for colon carcinoma. This may, in part, be a consequence of the diversified function of the normal human colon. This study addresses the possibility that the expression of urokinase and its receptor is inversely related to differentiation in colon carcinoma.

Six colon carcinoma cell lines including three well-differentiated (CBS, GEO, FET) and three poorly differentiated ones (HCT116, HCT116b, RKO) were screened for urokinase receptor display and secretion of the plasminogen activator. A radioreceptor assay was used to determine receptor levels. Binding of radioactive urokinase to colon cells was saturable, specific, and time dependent. Cell-bound ¹²⁵I-labeled protease was unaffected by the presence of epidermal growth factor, low-molecular-weight urokinase, plasminogen, or transferrin. Time course studies revealed that maximum amounts of radioactive tracer were bound in a 30-min period with no change occurring over the course of a 90-min incubation. Scatchard analysis of ligand binding indicated that the well-and poorly differentiated cells could be separated on the basis of receptor display; the aggressive RKO, HCT116, and HCT116b expressed in excess of 10⁵ sites per cell, while the more indolent CBS, GEO, and FET possessed less than 1.5 x 10⁴ receptors per cell.

The colon carcinoma cells were also analyzed for urokinase in the conditioned medium. Low levels of the plasminogen activator (0.8 to 1.3 ng/ml/10⁶ cells/72 h) were associated with the more "mature" cells. This was in contrast to the elevated levels of the protease (3.9 to 11.4 ng/ml/10⁶ cells/72 h) present in the medium derived from the more aggressive cells (HCT 116, HCT116b, RKO). Thus, secreted urokinase and/or the expression of cellular receptor for the plasminogen activator may provide useful measurements of the degree of undifferentiation of in vitro colon carcinoma.

¹ This work was supported by NIH Grant CA 34432.

² To whom requests for reprints should be addressed.

Received 9/ 8/87. Revised 1/11/88. Accepted 3/ 4/88.

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