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Methotrexate polyglutamates in cultured human cells
Abstract:
Cultured human fibroblasts accumulated methotrexate polyglutamates to levels far in excess of the dihydrofolate reductase binding capacity. After four days in methotrexate-free medium the intracellular drug level dropped by 70% but nearly 80% of the remaining methotrexate was in the form of polyglutamates. Reduced folates prevented the accumulation of polyglutamates and the effects of methotrexate on deoxyuridine incorporation into DNA and cell growth if present along with methotrexate from the beginning of the incubation. However, the reduced folates were less effective if added to cells after a long exposure to methotrexate alone. Thymidine, glycine, and adenosine (GAT) prevent methotrexate toxicity only if maintained in the incubation medium. However, preincubation with methotrexate and GAT permits continued synthesis and accumulation of polyglutamates so that when the GAT and methotrexate were removed, toxicity from the retained methotrexate polyglutamates could be expressed. (2,4-diamino-5-(3'4'-dichlorophenyl)-6 methyl pyrimidine (DDMP), an antifol that does not form polyglutamate derivatives, inhibited deoxyuridine incorporation into DNA as long as the DDMP remained in the culture medium. Compared to what was seen with longer exposures to methotrexate, removal of DDMP resulted in a greater reversal of the inhibition of deoxyuridine incorporation.
Insights
Methotrexate forms polyglutamates in cells, persisting even after drug removal. These polyglutamates contribute to toxicity, and their formation can be influenced by reduced folates and other compounds.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Methotrexate (MTX) is a chemotherapy drug that inhibits dihydrofolate reductase.
- Intracellular accumulation of MTX and its metabolites, particularly polyglutamates, influences its efficacy and toxicity.
- Understanding the dynamics of MTX polyglutamate formation and clearance is crucial for optimizing cancer therapy.
Purpose of the Study:
- To investigate the accumulation and persistence of methotrexate polyglutamates in cultured human fibroblasts.
- To examine the impact of reduced folates and other compounds on MTX polyglutamate formation and MTX-induced toxicity.
- To compare the effects of MTX polyglutamates with antifolates that do not form polyglutamates.
Main Methods:
- Culturing human fibroblasts and exposing them to methotrexate.
- Measuring intracellular drug levels and the proportion of methotrexate polyglutamates.
- Assessing the effects of reduced folates and thymidine, glycine, and adenosine (GAT) on MTX accumulation and toxicity.
- Evaluating the impact of 2,4-diamino-5-(3',4'-dichlorophenyl)-6 methyl pyrimidine (DDMP) on deoxyuridine incorporation.
Main Results:
- Fibroblasts accumulated MTX polyglutamates exceeding dihydrofolate reductase binding capacity.
- MTX polyglutamates persisted intracellularly for days after MTX removal, contributing to ongoing toxicity.
- Reduced folates prevented polyglutamate accumulation and MTX effects when added early, but were less effective after prolonged MTX exposure.
- GAT prevented MTX toxicity only when continuously present; preincubation allowed polyglutamate accumulation and subsequent toxicity.
- DDMP, an antifol that doesn't form polyglutamates, inhibited DNA synthesis only while present, with greater reversal upon removal compared to MTX.
Conclusions:
- Methotrexate polyglutamates are long-lived intracellular metabolites that contribute significantly to MTX toxicity.
- The formation and persistence of MTX polyglutamates are influenced by the presence of reduced folates and other compounds.
- Antifolates like DDMP, which do not form polyglutamates, offer a different toxicity profile with more readily reversible effects upon removal.