Cell Lines Mechanisms of Doxorubicin Resistance in Two Human Tumor Pharmacological and Biological Evidence for Differing

The cellular pharmacologyof doxorubicin resistance (DOXR)has most commonly been associated with decreased drug uptake, enhanced drug efflux, cross-resistance to multiple anticancer agents, and the overpro ductionof a M, 170,000 cell surface glycoprotein (termed P-glycoprotein). In this study, the pharmacological and genetic characteristics of two newly derived human DOX" sublines were examined. These DOX" sublines were established following continuously increasing DOX expo sure until a 222-fold resistant fibrosarcoma subline (HT1080/DR4) and a 285-fold resistant colon adenocarcinoma subline (LoVo/DRS) were developed. However, three major lines of evidence suggest that despite the similar selection strategy, the mechanism of DOX" differs signifi cantly between these two cell lines. First, Western blotting using the C219 antibody specific to P-glycoprotein revealed the overexpression of the M, 170,000 cell surface glycoprotein in LoVo DOX" cells but not in HT1080 DOX" cells. Second, LoVo DOXR cells are cross-resistant to vincristine, actinomycin D, colchicine, etoposide, and gramicidin D, but not to 1-iS-D-arabinofuranosylcytosine.In contrast, HT1080 DOXRcells display cross-resistance to vincristine, actinomycin D, vinblastine, and etoposide; however, they are not cross-resistant to gramicidin D, and show an increased (~18-fold) cross-resistance to 1-0-D-arabinofuranosylcytosine. Third, intracellular DOX accumulation (as measured by [14C]DOXat 1-h and high-performance liquid chromatography analysis) was decreased ~2.7-fold in LoVo DOX" cells and ~2.0-fold in HT1080/ DR4 cells. However, while net accumulation studies in the presence of 5 Mg/mlverapamil reversed DOXR to parental values in LoVo colon ade nocarcinomacells, it only minimally decreased DOX resistance (12.6%) in HT1080/DR4 cells. Efflux patterns of |'4C|DOX were similar for the DOXRsublines with an -50% decrease in DOX retention after l h when compared to their respective parental cell lines. Our results suggest that DOX" in LoVo/DR5 cells may result from overexpression of P-glycopro tein. In contrast, DOX" in HT1080/DR4 appears to be non-P-glycoprotein mediated and may be related to an alternative mechanism capable of altering drug efflux or differential drug binding.