Detection of Thymidylate Synthase Modulators by a Novel Screening Assay
- Grace Cancer Drug Center, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York
- Dr. Bruce J. Dolnick, Grace Cancer Drug Center, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. E-mail: bruce.dolnick{at}roswellpark.org.
Abstract
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.
Footnotes
-
This study was supported by United States Public Health Service grants CA91114 and CA86876 (to B.J.D.) and Roswell Park Cancer Institute Core grant CA16056.
- Abbreviations:
- TS
- thymidylate synthase
- TBE
- thymidylate synthase-specific binding elements
- nt
- nucleotides
- EGR-I
- early growth response gene
- 5-FU
- 5-fluorouracil
- FUdR
- 5-fluoro-2′-deoxyuridine
- TFT
- trifluorothymidine
- Act D
- actinomycin D
- AC
- 5-azacytidine
- TG
- 8-thioguanosine
- FUrd
- 5-fluorouridine
- MTX
- methotrexate
- bp
- base pair(s)
- FBS
- fetal bovine serum
- G418
- geneticin
- RLU
- relative light units
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- FdUMP
- 5-fluorodeoxyuridylate
- AG331
- N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino-benz[c,d]indole glucuronate
- AG337
- nolatrexed
- ZD1694
- raltitrexed (Tomudex)
-
- Received July 30, 2002.
- Accepted October 9, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
