title: The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cells.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine, National Defense Medical College, Tokorozawa, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Takemura
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: W
authors.initials: W
authors.lastname: Gibson
authors.forename: R
authors.initials: R
authors.lastname: Kimbell
authors.forename: H
authors.initials: H
authors.lastname: Miyachi
authors.forename: A L
authors.initials: AL
authors.lastname: Jackman
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D004351:Drug Resistance; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000201:enzymology; D008727:Methotrexate / Q000494:pharmacology*; D010453:Peptide Synthases / Q000378:metabolism; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D011624:Pteroylpolyglutamic Acids; D011799:Quinazolines; D012333:RNA, Messenger; D013876:Thiophenes; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011623:gamma-Glutamyl Hydrolase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; C068874:raltitrexed; D008727:Methotrexate