Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines

Abstract

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.