The copper export pump ATP7B modulates the cellular pharmacology of carboplatin in ovarian carcinoma cells.

Human tumor cells lines with acquired resistance to cisplatin (DDP) and carboplatin (CBDCA) are often cross-resistant to copper and vice versa, and some DDP-resistant cells overexpress the copper export pump ATP7B. We sought to demonstrate that ATP7B directly mediates resistance to DDP and CBDCA by stably transfecting human carcinoma cells with a vector designed to express ATP7B. Increased expression of ATP7B rendered all three cell lines tested more resistant to a 1-h exposure to DDP (1.6-2.6-fold), CBDCA (1.5-1.6-fold), and copper (1.2-1.4-fold). The effect of ATP7B on the cellular pharmacology of 64Cu and [14C]CBDCA was investigated in more detail using one cell pair (2008 cells transfected with an empty vector or an ATP7B-expressing vector). In the 2008/ATP7B subline, steady-state copper levels were decreased under both basal and copper-supplemented conditions, as was steady-state CBDCA content upon exposure to 50 microM [14C]CBDCA. Over the first 5 min, the average rate of accumulation of copper and CBCDA in the 2008/ATP7B cells was reduced by 37 and 61%, respectively. Efflux was more rapid from 2008/ATP7B cells for both copper and CBDCA. Two-compartment modeling indicated that the second phase of efflux was increased by a factor of 3.9-fold for CBCDA and to an even greater extent for copper. We conclude that expression of ATP7B regulates sensitivity to CBDCA as well as to DDP and copper and that a transporter that normally mediates copper homeostasis modulates the cellular pharmacology of CBDCA.

Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper.

Impaired uptake of cisplatin (DDP) consistently accompanies the acquisition of resistance to the platinum drugs. The pathways by which DDP enters or exits from cells remain poorly defined. Using three pairs of human ovarian carcinoma cell lines, each consisting of a sensitive parental line and a stably DDP-resistant subline derived by in vitro selection, resistance to DDP was found to be accompanied by cross-resistance to Cu. Accumulation of DDP in the resistant sublines ranged from 38 to 67% of that in the parental line at 1 h, and DNA adduct formation varied from 10 to 38% of that in the sensitive cells. The DDP-resistant cells had 22-56% lower basal levels of copper, and the copper levels were only 27-46% of those observed in the sensitive parental lines after a 24-h exposure to medium supplemented with copper. The initial influx rate for DDP in the three resistant cell lines ranged from 23 to 55% of that in the sensitive cells of each pair; the initial influx rate for copper in the resistant cells varied from 56 to 75% of control. Studies performed using one pair of cell lines demonstrated that for both copper and DDP the initial efflux rate was lower, whereas the terminal efflux rate was higher in the resistant cells. On Western blot analysis all three resistant lines exhibited increased expression of one or the other of the two copper export pumps (ATP7A or ATP7B) with no change in the HAH1 chaperone. We conclude that the acquisition of DDP resistance in ovarian carcinoma is accompanied by alterations in the cellular pharmacology of DDP that are paralleled by similar changes in the uptake and efflux of copper. These results are consistent with the concept that DDP enters and exits from the cell via transporters that normally mediate copper homeostasis.