Detection of the 170 kDa P-Glycoprotein in Neoplastic and Normal Tissues

Abstract

A membrane purification procedure and an immunoblotting assay have been designed to allow screening of human solid tumors for overexpression of the GP170 glycoprotein without employing a disaggregation method to obtain cell suspensions. The electrophoresed membrane proteins were probed, after Western Blotting, with the C219 monoclonal antibody and iodinated Protein A. The labeling intensity of the bands on the autoradioimmunoblots were quantified by densitometry. To test for the presence of GP170, we used membranes from the UV 2237 fibrosarcoma line and its adriamycin-resistant variant ADM^R, grown in vitro or as solid tumor in mice. Membranes of human normal and tumor tissues obtained from previously untreated patients were also tested. An immunoreaction was observed in the adriamycin-resistant UV 2237 lines grown in vitro or in vivo. Quantitatively, the binding of the resistant cell line grown in vitro was higher than that observed in cells grown in mice. Bands in the GP 170 region were observed in 4/7 normal and in 7/7 tumor colon tissues and in the normal medulla from 2 patients with cancer of the renal cortex. No reaction could be found in samples from normal tissue, primary tumor or nodal metastasis from 7 patients with breast cancer.