Membrane Vesicle Formation Due to Acquired Mitoxantrone Resistance in Human Gastric Carcinoma Cell Line EPG85-2571

A newly established gastric carcinoma cell line (EPG85-257P) exhib ited a high sensitivity to mitoxantrone (DHAD) as determined by a monolayer proliferation assay. The concentration to inhibit cell growth to 50% of controls (IG»)was 0.0022 «¿g/ml culture medium. The cells were continuously incubated for more than 4 months in the presence of stepwise increased concentrations of DHAD, and the ICS) was increased to 0.41 Mg/ml,i.e., 186.4-fold. This resistant variant was named EPG85257RNOV. The EPG85-257RNOV cells became cross-resistant to Adriamycin with enhanced K ..„ by 10.5-fold and to daunomycin with enhanced IC»by 3.9-fold. No distinct resistance was observed to vinblastine, vincristine, and colchicine. Verapamil (!()''. 4 x 10 " and Id \i) and cyclosporin A (10"*, 3 x 10"* and IO"5 M) did not reverse DHAD resistance. As shown by immunocytochemistry (monoclonal antibodies: C219 and JSB-1) and Northern blot analysis, DHAD resistance was not associated with the appearance of the multidrug resistance (MDR)associated (M, 170,000) P-glycoprotein or the overexpression of Pglycoprotein mRNA. The data indicate a chemoresistance pattern unlike typical MDR (often called "atypical" MDR). The phenotypes of parent and resistant EPG85-257 cells were com pared using interference contrast microscopy, electron microscopy, and immunocytochemistry. After DHAD application the following structural characteristics were found to be associated with emergence of resistance: (a) intensive formation of surface vesicles in the resistant variant. Such vesicles were almost absent in sensitive cells; (b) the vesicles contained the selecting DHAD which was visualized by its blue color; and (<•) in electron microscopy the vesicles were formed by an inner and an outer double membrane, presumably derived from the plasmalemma. These observations suggest a complex cellular mechanism responsible for DHAD resistance which includes formation of membrane vesicles, vesic ular drug binding, and drug compartmentalization.