The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cells.

The influence of drug exposure conditions on the development of resistance to methotrexate (MTX) or ZD1694 was studied by treating MOLT-3 human lymphoblastic-leukaemia cells in a continuous or a pulsatile (high-dose, short term) drug-exposure schedule. Continuous exposure of the cells to MTX with stepwise escalation of the drug concentrations resulted in a MTX-resistant sub-line (MOLT-3/MTX(10000)) with impaired reduced-folate carrier (RFC) and increased dihydro-folate-reductase (DHFR) activity. Conversely, a MTX-resistant clone (MOLT-3/MTX. P-9) with unaltered RFC and DHFR activity, but with decreased cellular accumulation of anti-folates, was selected by high-dose short-term treatment of the cells with MTX. MTX resistance in the latter cells was pronounced after short-term rather than continuous-exposure incubation with MTX, suggesting defective polyglutamation of the drug. On the other hand, 2 ZD1694-resistant sub-lines which were established by continuous (MOLT-3/ZD1694. C) or by pulsatile drug-exposure schedule (MOLT-3/ZD1694.P-9) demonstrated extremely low accumulation and poor retention of [3H]ZD1694, with no change in initial drug uptake and little or no increase of thymidylate-synthase (TS) activity irrespective of drug exposure conditions for their establishment. HPLC analysis displayed a virtual absence of ZD1694 polyglutamates in both ZD1694-resistant sub-lines and low accumulation in MOLT-3/MTX.p-9 as compared to the parent line. However, folylpolyglutamate-synthetase(FPGS) mRNA was only moderately decreased in the 2 ZD1694-resistant sub-lines and to an even lesser extent in MOLT-3/MTX.p-9. In addition, gamma-glutamyl-hydrolase(GGH) activity was not increased, but was slightly down-regulated in the polyglutamation-defective sub-lines. These results indicate that the mechanism(s) of the resistance developed may depend not only on drug-exposure conditions while raising resistance but also on the biochemical properties of the drug.