EFFECT OF METHOTREXATE ON MURINE BONE-MARROW CELLS IN-VITRO - EVIDENCE OF A REVERSIBLE ANTIPROLIFERATIVE ACTION

Methotrexate (MTX) acts by inducing cellular depletion of reduced fola tes, which ultimately leads to an inhibition of DNA synthesis. Like ma ny anticancer drugs, this antimetabolite has little selectivity for tu mor cells, and its effectiveness is limited by toxicity to normal tiss ues, particularly gastrointestinal epithelium and bone marrow. Previou s studies have shown that MTX inhibits colony formation of the hematop oietic progenitor cells (CFU-C) in vitro. Whether this effect is due t o a cytotoxic or a cytostatic mechanism has not been resolved. The pre sent study was undertaken to eludicate the mechanism by which MTX inhi bits CFU-C formation. Bone marrow cells in agarose cultures supplement ed with recombinant murine granulocyte-macrophage colony-stimulating f actor (rmGM-CSF) were incubated for 7 days in the presence or absence of MTX. Exposure to 33 nM to 1 mu M MTX reduced colony formation by mo re than 80% when compared to control cultures. When bone marrow suspen sion cultures supplemented with rmGM-CSF were incubated for 5 days in the presence or absence of MTX, exposure to 10 nM to 1 mu M MTX result ed in a 60 to 80% reduction in cell numbers when compared to untreated cultures. Residual CFU-C numbers were determined in the same cultures by replating into agarose. Exposure to 10 nM MTX was found to enhance CFU-C recovery three-fold as compared to controls and cultures expose d to higher MTX concentrations. Addition of 10 mu M of the reduced fol ate leucovorin (LV; 5-formyl-tetrahydrofolate) prevented CFU-C accumul ation in the presence of 10 nM MTX. The kinetics of LV rescue of CFU-C , pre-exposed to 100 nM MTX, were investigated in clonogenic assays. T he addition of 1 mu M LV to semisolid bone marrow cultures preincubate d with 100 nM MTX for up to 8 days completely abolished the inhibition of colony formation seen with 100 nM MTX alone. When the dose range o f MTX was expanded from 33 nM to 3.3 mu M, we found that administratio n of 10 mu M LV on day 5 rescued the hematopoietic progenitors from MT X inhibition in all groups. These observations suggest that MTX is not cytotoxic to hematopoietic progenitors over its entire dose range but that it can induce a reversible block in the proliferation and differ entiation of cells in the progenitor compartment.