Amplification of the thymidylate synthase gene in anN 10-propargyl-5,8-dideazafolic-acid-resistant human leukemia,MOLT-3 cell line developed in pteroylglutamic acid,but not in leucovorin |
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Authors: | Hayato Miyachi Yuzuru Takemura Hiroyuki Kobayashi Yasuhiko Ando |
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Affiliation: | (1) Department of Clinical Pathology, Tokai University School of Medicine, Bohseidai, 259-11 Isehara, Kanagawa, Japan;(2) Department of Laboratory Medicine, National Defense Medical College, 3-2 Namiki, 359 Tokorozawa, Saitama, Japan |
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Abstract: | The types of folates used during the development of resistance to methotrexate have been suggested to play an important role in the mechanisms of established resistance. In this study, effects of reduced and oxidized folates on the development of resistance to a thymidylate synthase (TS) inhibitor,N10-propargyl-5,8-dideazafolic acid (CB3717), were exmined in the human leukemia cell line MOLT-3. MOLT-3 cells were made resistant to CB3717 by soft-agar cloning in RPMI-1640 medium with either pteroylglutamic acid (PGA) or a more physiological folate (10 nM leucovorin). A 40-fold CB3717-resistant subline developed in PGA (MOLT-3/CB371740-PGA) showed amplification of the TS gene with a concomitant increased level in the gene expression. A 200-fold CB3717-resistant subline (MOLT-3/CB3717200-PGA), which was derived from MOLT-3/CB371740-PGA, showed further enhancement of amplification of the TS gene. In contrast, even a 200-fold CB3717-resistant subline developed in leucovorin (MOLT-3/CB3717200-LV) showed neither amplification nor overexpression of the TS gene. Both MOLT-3/CB3717200-PGA and MOLT-3/CB3717200-LV cells showed decreased membrane transport of PGA as well as methotrexate. These results suggest that the types of folates used during the development of CB3717 resistance may play a role in resistance, and that impaired transport of PGA, in CB3717-resistant MOLT-3 cells developed in PGA, might have accelerated amplification of the TS gene.Abbreviations CB3717 N10-propargyl-5,8-dideazafolic acid - DHFR dihydrofolate reductase - FCS fetal calf serum - IC50 50% inhibitory concentration for cell growth by MTT assay - LV leucovorin, 5-formyltet-rahydrofolate - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) - PGA pteroylglutamic acid - TS thymidylate synthase |
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Keywords: | N10-propargyl-5,8-dideazafolic acid Thymidylate synthase Gene amplification Folate |
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