Polyglutamate Formation Research Articles

SYNTHESIS OF METHOTREXATE POLYGLUTAMATES IN CULTURED HUMAN CELLS

The pteroylglutamate analog methotrexate (MTX) is a potent inhibitor of the enzyme dihydrofolate reductase and an important antineoplastic agent. Recently synthesis of poly-y-glutamyl metabolites of MTX has been reported in both mice and rats. Cultured human fibroblasts were incubated with purified tritium-labeled MTX and Sephadex G-15 gel chromatography was used to study the formation of these MTX polyglutamates. Synthesis of polyglutamates occurred sequentially and was dependent on the concentration of MTX in the culture medium, duration of incubation, and stage of the culture cycle. After incubation with 0.1 μM MTX the cells accumulated methotrexate monoglutamate [MTX(G1)] and diglutamate [MTX(G2)] so that by 24 hours they represented the majority of labeled MTX in the fibroblasts. After 4 hours MTX level remained relatively stable and further increase in labeling was due to accumulation of polyglutamates. When tritiated MTX was removed from the culture medium at 24 hours, no change in the proportion of MTX and MTX polyglutamates occurred over the next 24 hours. However when triated MTX was replaced after 24 hours with equimolar cold MTX, rapid loss of labeled MTX occurred with sequential formation and loss of methotrexate polyglutamates. These results suggest that like the folate polyglutamates, MTX polyglutamates may represent major active compounds.

Synthesis of folate polyglutamates in human cells.

1. Synthesis of polyglutamate derivatives from radioactively labelled folic acid, folinic acid and methotrexate has been studied in human phytohaemagglutinin-transformed lymphocytes. 2. With labelled folic acid as the precursor, approximately 20% of the labelled cell folate was found in each of the mono-, tetra-, penta- and hexaglutamate peaks after 72 h of incubation. At earlier times, the amounts of labelled folate polyglutamates were proportionately greater and the amount of labelled higher folate polyglutamates was lower. After only 4 h incubation over 80% of the intracellular labelled folate was still in the monoglutamate form. 3. With labelled folinic acid used as precursor, approximately 30% of labelled cell folate after 72 h incubation was in the form of folate pentaglutamate, with tri- and tetra-glutamate forming the other major peaks. The speed of formation of polyglutamate derivatives was substantially more rapid than from folic acid. 4. Methotrexate was found to decrease considerably the amount of folate polyglutamate formation from labelled folic acid in lymphocytes byt nevertheless some di-, tri- and traces of tetra-glutamate were formed. On the other hand, methotrexate had no effect on folate polyglutamate formation with labelled folinic acid used as the precusor. 5. Polyglutamate derivatives of labelled methotrexate were formed by human lymphocytes with mean amounts of 4-5% of di-, 1-5% of tri-, 1-0% of tetra- and 0-7% of penta-glutamate derivatives being formed over the period 48-72h of culture. 6. Pentaglutamate derivatives probably constitute the largest group of intracellular folates in human cells but a complex mixture exists. 7. The enzyme synthesizing polyglutamate derivatives of folate in human cells prefers a reduced folate as substrate but the requirement for a reduced form is not absolute. The nature of the reduced folate is uncertain but it is suggested on the basis of previous work that tetrahydrofolate rather than methylhydropfolate or any other reduced folate monoglutamate is the preferred substrate.