Methotrexate cross-resistance in a mitoxantrone-selected multidrug-resistant MCF7 breast cancer cell line is attributable to enhanced energy-dependent drug efflux

Cellular resistance to the antifolate methotrexate (MTX) is often caused by target amplification, uptake defects, or alterations in polyglutamylation. Here we have examined MTX cross-resistance in a human breast carcinoma cel l line (MCF7/MX) selected in the presence of mitoxantrone, an anticancer ag ent associated with the multidrug resistance (MDR) phenotype. Examination o f protein expression and enzyme activities shea ed that MCF7/MX cells displ ayed none of the classical mechanisms of MTX resistance. They did, however, exhibit an ATP-sensitive accumulation defect accompanied by reduced polygl utamylation. Although the kinetics of drug uptake was similar between paren tal and resistant cells, the resistant cells exhibited increased energy-dep endent drug efflux. This suggested the involvement of an ATP-binding casset te (ABC) transporter. However, cells transfected with the breast cancer res istance protein (BCRP)-the ABC transporter known to be highly overexpressed in MCF7/MX cells and to confer mitoxantrone resistance (D. D. Ross et al, J, Natl. Cancer Inst. 91: 429-433, 1999)-were not MTX resistant, which sugg ested that this transporter is not involved in MTX cross-resistance. Moreov er, members of the MRP protein family of transport proteins, which had prev iously been implicated in MTX resistance, were not found to be overexpresse d in the MCF7/MX cells, Thus, our data suggest that a novel MTX-specific ef flux pump may be involved in this unusual cross-resistance phenotype.