Detection of thymidylate synthase modulators by a novel screening assay.
Thymidylate synthase (TS), a key cancer chemotherapeutic target, catalyzes the conversion of deoxyuridylate to thymidylate. TS can serve as a repressor of its own synthesis by binding to its own mRNA through TS-specific binding elements (TBEs). In this report, we describe the use of a luciferase reporter plasmid containing two TBEs that can be used as a tool for the identification and initial profiling of compounds that modulate TS activity, levels, or ability to bind mRNA. To validate this model, we evaluated several groups of drugs. Thus, cells were exposed to the pyrimidine analogs 5-fluorouracil (5-FU), 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FUdR), trifluorothymidine (TFT); to the nonpyrimidine TS-inhibitors AG-331, nolatrexed (AG337), and raltitrexed (ZD1694); or to drugs with other primary sites of action (methotrexate, actinomycin D, 5-azacytidine, 8-thioguanosine). Except for 5-azacytidine and 8-thioguanosine, all compounds examined induced luciferase activity compared with untreated cells. Effects of luciferase activity inducing drugs through TS-affected translation were confirmed by examinations of TS protein and mRNA levels. Treatment of H630-C6 cells with 5-FU, FUrd, FUdR, TFT, AG331, AG337, ZD1694, and methotrexate up-regulated TS levels as determined by Western blot analysis, although TS mRNA levels remained unchanged as determined by reverse transcription-polymerase chain reaction. Our studies demonstrate a novel application of a TBE-dependent reporter plasmid that could be used for the high-throughput identification of potential chemotherapeutic agents that modulate TS RNA-binding activity, either directly or indirectly.
Wu
Qi
Q
Grace Cancer Drug Center, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Dolnick
Bruce J
BJ
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D051766:Early Growth Response Protein 1; D004790:Enzyme Induction / Q000187:drug effects; D005467:Floxuridine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D017874:Immediate-Early Proteins; D008156:Luciferases / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D005467:Floxuridine; D008156:Luciferases; D013940:Thymidylate Synthase; D005472:Fluorouracil