Molecular characterization of human acute leukemia cell line resistant to ZD9331, a non-polyglutamatable thymidylate synthase inhibitor.

Abstract

ZD9331 is a non-polyglutamatable, potent quinazoline antifolate inhibitor of thymidylate synthase (TS). In an effort to clarify the exact mechanism of resistance to this novel TS inhibitor, we examined the molecular alterations in its target enzyme TS, the transport protein (reduced folate carrier, RFC), and folylpolyglutamate synthetase (FPGS) in a human acute lymphoblastic leukemia cell line, MOLT-3, made resistant to ZD9331. A 310-fold resistant subline was established after 6 months exposure to the drug at concentrations up to 7 microM, and was designated MOLT-3/ ZD9331. MOLT-3/ZD9331 showed crossresistance to CB3717 (4.8-fold), raltitrexed (63-fold) and methotrexate (MTX) (120-fold), but retained sensitivity to trimetrexate (0.88-fold). The resistant cells demonstrated impaired initial cellular uptake and low accumulation of [3H]MTX in accordance with a decreased expression of RFC1, suggesting the downregulation of RFC. However, Southern blot analysis demonstrated no change in gene copy number nor gross rearrangement of RFC1 in the resistant cells. In addition, MOLT-3/ZD9331 showed amplification of the TS gene with a concomitantly increased level in the gene expression. In contrast, the expression of FPGS did not alter. These results demonstrate that continuous exposure of the cells to ZD9331 leads not only to a decreased expression of RFC1 but also to TS gene amplification and overexpression. The resistant mechanisms are likely to be regulated both at a genetic and a transcriptional level for different resistance phenotypes in the ZD9331-resistant MOLT-3 cells.