Methotrexate (MTX)-resistant sublines of malignant human cells were selected in vitro by stepwise increase in
drug concentration in the medium. By this procedure a subline of
Burkitt's lymphoma cells (RAJI) was made 290-fold resistant (RAJI/MTX-R),
T-cell leukemia cells (CCRF-CEM) were obtained 210-fold resistant (CEM/MTX-R), and 3 MTX-resistant human
osteosarcoma lines were selected: TE-85/MTX-R (19-fold resistant; relative to wild-type); MG-63/MTX-R (8-fold resistant); and SAOS-2/MTX-R (200-fold resistant). We also studied a B-cell lymphoblastoid line, WI-L2/m4, that was 13,000-fold resistant. Assay of cellular
dihydrofolate reductase (DHFR) showed the following pattern of activity in resistant cell lines, relative to parental cell activity: RAJI/MTX-R, 550-fold increased; CEM/MTX-R, unchanged; TE-85/MTX-R, 4-fold increased; MG-63/MTX-R, 6-fold increased; SAOS-2/MTX-R, unchanged; and WI-L2/m4, 110-fold increased. Measurement of MTX membrane transport showed decreased uptake in CEM/MTX-R and SAOS-2/MTX-R, relative to parental cell lines. The other DHFR-overproducing cells all gave normal initial MTX uptake rates but increased total uptake. The DHFR-overproducing lines all had significant cross-resistance to both
metoprine and
trimetrexate; the two lines with defective MTX transport were not cross-resistant, and the CEM/MTX-R cells showed collateral sensitivity to these agents. Only minor cross-resistance to
homofolic acid was found in all MTX-resistant lines. The highly MTX-resistant RAJI/MTX-R and WI-L2/m4 cells showed minor cross-resistance to the dual inhibitor of
thymidylate synthetase and DHFR,
CB3717 (5- and 15-fold, respectively). These studies demonstrated that, depending upon the mechanism of resistance, MTX-resistant human
tumor cells may be effectively killed by
antifolates with different routes of uptake into cells, or with a different
enzyme target. Thus, there are at least three functionally distinct classes of
folate antagonist with antitumor activity.