Role of methotrexate polyglutamates in methotrexate- and sequential methotrexate-5-fluorouracil-mediated cell kill.
The synthesis of methotrexate (MTX) polyglutamates has been investigated in a human leukemia cell line, CCRF-CEM. An increase in either the extracellular MTX concentration (0.1-10 microM) or exposure time (4-24 h) allowed a greater accumulation of polyglutamate derivatives. The species which predominated (di-, tri-, or tetraglutamate) was also time and concentration dependent. Derivatives up to the pentaglutamate were readily detectable, with hexaglutamate present under some conditions. Polyglutamate derivatives were preferentially retained when the cells were transferred to drug-free medium. MTX itself rapidly exited the cells under these conditions. In the absence of extracellular MTX, the performed intracellular MTX polyglutamates continued to be elongated. Accumulation of MTX polyglutamates, particularly triglutamate and longer species, positively correlated with cytotoxicity to CCRF-CEM cells as measured by a clonogenic assay. Investigation of the methotrexate-fluorouracil interaction revealed no effect of 5-fluorouracil on MTX polyglutamate accumulation under various conditions where widely different degrees of cytotoxicity were found. The synergy observed when MTX precedes fluorouracil treatment is therefore not an effect on MTX polyglutamylation. MTX polyglutamates may, however, play an indirect role in this synergy.
J J
JJ
McGuire
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E
Mini
P
P
Hsieh
J R
JR
Bertino
D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D005472:Fluorouracil / Q000633:toxicity*; D006801:Humans; D007942:Leukemia, Experimental / Q000188:drug therapy / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D010455:Peptides / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism* / Q000633:toxicity
D010455:Peptides; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D005472:Fluorouracil; D008727:Methotrexate