Resistance to Tomudex (ZD1694): Multifactorial in human breast and colon carcinoma cell lines

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 NCl H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 mu M, resulted in stably resistant cell lines (MCF(TDX) and H630(TDX)) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCF(TDX) and a 52-fold increase in TS levels in H630(TDX) cell lines. Despite continued exposure of these cells to 2.0 mu M TDX, TS protein and TS mRNA expression decreased to parental levels in H630(TDX) cells, whereas in MCF(TDX) cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCF(TDX) cell line. TDX uptake was 2-fold higher in resistant MCF(TDX) cells than in parental MCF-7 cells, whereas in H630(TDX) 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 H630(TDX) cells was noted when compared with the H630 parental cells. In H630(TDX) cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630(TDX) 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 MCF(TDX) and H630(TDX) 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.