EVIDENCE FOR THE INVOLVEMENT OF 2 DISTINCT MEMBRANE-PROTEINS IN ADRIAMYCIN RESISTANCE IN CHINESE-HAMSTER LUNG-CELLS

Abstract

Chinese hamster lung cells resistant to Adriamycin were labeled with inorganic [32P]orthophosphate and thereafter incubated with low levels of N-ethylmaleimide. Plasma membranes and endoplasmic reticulum were isolated and the phosphorylated proteins were analyzed after polyacrylamide gel electrophoresis. The results demonstrate that both plasma membranes and endoplasmic reticulum from resistant cells contain two highly phosphorylated proteins [Mr 180,000 (p180) and Mr 220,000 (p220)] which are present in very low levels in these membrane fractions prepared from drug sensitive cells. p220 is present in much higher levels in the endoplasmic reticulum as compared to the plasma membranes whereas p180 is equally distributed in these two membrane fractions. When resistant cells revert to drug sensitivity there is a parallel loss in the phosphorylation levels of p180 and p220. Labeling of membrane proteins with 125I in the presence of chloramine-T also reveals that p180 and p220 are present in significantly greater levels is resistant membranes as compared to similar fractions prepared from drug sensitive cells. Partial digests of phosphorylated p180 and p220 produced with chymotrypsin or V8 protease reveal that each protein has a distinct phosphopeptide pattern. Both p180 and p220 are phosphorylated exclusively at serine residues. The results of this study therefore suggest that resistance to Adriamycin in Chinese hamster lung cells requires the involvement of two distinct proteins which are both bound to cell membranes.