A. Stromhaug et al., EFFECT OF METHOTREXATE ON MURINE BONE-MARROW CELLS IN-VITRO - EVIDENCE OF A REVERSIBLE ANTIPROLIFERATIVE ACTION, Experimental hematology, 23(5), 1995, pp. 439-443
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.